InteractiveFly: GeneBrief

Odorant receptor co-receptor: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Odorant receptor co-receptor

Synonyms - Odorant receptor 83b

Cytological map position - 83A2

Function - G-protein coupled receptor

Keywords - odorant receptor, seven-transmembrane proteins

Symbol - Orco

FlyBase ID: FBgn0037324

Genetic map position - 3R

Classification - odorant receptor

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Del Pino, F., Jara, C., Pino, L., Medina-Muñoz, M.C., Alvarez, E. and Godoy-Herrera, R. (2015). The identification of congeners and aliens by Drosophila larvae. PLoS One 10: e0136363. PubMed ID: 26313007
Summary:
This study investigated the role of Drosophila larva olfactory system in identification of congeners and aliens. These activities are important in larva navigation across substrates, and have implications for allocation of space and food among species of similar ecologies. Wild type larvae of cosmopolitan D. melanogaster and endemic D. pavani, which cohabit the same breeding sites, use species-specific volatiles to identify conspecifics and aliens moving toward larvae of their species. D. gaucha larvae, a sibling species of D. pavani that is ecologically isolated from D. melanogaster, does not respond to melanogaster odor cues. Similar to D. pavani larvae, the navigation of pavani female x gaucha male hybrids is influenced by conspecific and alien odors, whereas gaucha female x pavani male hybrid larvae exhibit behavior similar to the D. gaucha parent. The two sibling species exhibit substantial evolutionary divergence in processing the odor inputs necessary to identify conspecifics. Orco (Or83b) mutant larvae of D. melanogaster, which exhibit a loss of sense of smell, do not distinguish conspecific from alien larvae, instead moving across the substrate. Syn97CS and rut larvae of D. melanogaster, which are unable to learn but can smell, move across the substrate as well. The Orco (Or83b), Syn97CS and rut loci are necessary to orient navigation by D. melanogaster larvae. Individuals of the Trana strain of D. melanogaster do not respond to conspecific and alien larval volatiles and therefore navigate randomly across the substrate. By contrast, larvae of the Til-Til strain uses larval volatiles to orient their movement. Natural populations of D. melanogaster may exhibit differences in identification of conspecific and alien larvae. Larval locomotion is not affected by the volatiles (Del Pino, 2015).
Bahk, S. and Jones, W. D. (2016). Insect odorant receptor trafficking requires calmodulin. BMC Biol 14: 83. PubMed ID: 27686128
Summary:
Like most animals, insects rely on their olfactory systems for finding food and mates and in avoiding noxious chemicals and predators. Most insect olfactory neurons express an odorant-specific odorant receptor (OR) along with Orco, the olfactory co-receptor. Orco binds ORs and permits their trafficking to the dendrites of antennal olfactory sensory neurons (OSNs), where together, they are suggested to form heteromeric ligand-gated non-selective cation channels. While most amino acid residues in Orco are well conserved across insect orders, one especially well-conserved region in Orco's second intracellular loop is a putative calmodulin (CaM) binding site (CBS). This study, exploreed the relationship between Orco and CaM in vivo in the olfactory neurons of Drosophila melanogaster. OSN-specific knock-down of CaM at the onset of OSN development was found to disrupt the spontaneous firing of OSNs and reduce Orco trafficking to the ciliated dendrites of OSNs without affecting their morphology. A series of Orco CBS mutant proteins was generated and found that none of them rescue the Orco-null Orco 1 mutant phenotype, which is characterized by an OR protein trafficking defect that blocks spontaneous and odorant-evoked OSN activity. In contrast to an identically constructed wild-type form of Orco that does rescue the Orco 1 phenotype, all the Orco CBS mutants remain stuck in the OSN soma, preventing even the smallest odorant-evoked response. Last, it was found that CaM's modulation of OR trafficking is dependent on activity. Knock-down of CaM in all Orco-positive OSNs after OR expression is well established has little effect on olfactory responsiveness alone. When combined with an extended exposure to odorant, however, this late-onset CaM knock-down significantly reduces both olfactory sensitivity and the trafficking of Orco only to the ciliated dendrites of OSNs that respond to the exposed odorant. In conclusion that study has show CaM regulates OR trafficking and olfactory responses in vivo in Drosophila olfactory neurons via a well-conserved binding site on the olfactory co-receptor Orco. As CaM's modulation of Orco seems to be dependent on activity, a model is proposed in which the CaM/Orco interaction allows insect OSNs to maintain appropriate dendritic levels of OR regardless of environmental odorant concentrations.
Retzke, T., Thoma, M., Hansson, B. S. and Knaden, M. (2017). Potencies of effector genes in silencing odor-guided behavior in Drosophila melanogaster. J Exp Biol [Epub ahead of print]. PubMed ID: 28235908
Summary:
The genetic toolbox in Drosophila offers a multitude of different effector constructs to silence neurons and neuron populations. This study investigated the potencies of several effector genes - when expressed in olfactory sensory neurons (OSNs) - to abolish odor-guided behavior in three different bioassays. Two of the tested effectors (tetanus toxin and Kir2.1) are capable of mimicking the Orco mutant phenotype in all of tested behavioral paradigms. In both cases the effectiveness depended on effector expression levels as full suppression of odor-guided behavior was observed only in flies homozygous for both Gal4-driver and UAS-effector constructs. Interestingly, the impact of the effector genes differed between chemotactic assays (i.e. the fly has to follow an odor gradient to localize the odor source) and anemotactic assays (i.e. the fly has to walk upwind after detecting an attractive odorant). In conclusion, these results underline the importance of performing appropriate control experiments when exploiting the Drosophila genetic toolbox and demonstrate that some odor-guided behaviors are more resistant to genetic perturbations than others.
Guo, H., Kunwar, K. and Smith, D. (2017). Odorant receptor sensitivity modulation in Drosophila. J Neurosci 37(39): 9465-9473. PubMed ID: 28871035
Summary:
The ability to modulate sensitivity in sensory systems is essential for useful information to be extracted from fluctuating stimuli in a wide range of background conditions. This study reveals that dephosphorylation of OrcoS289 that occurs upon prolonged odor exposure is a mechanism underlying reduction in odorant sensitivity in Drosophila primary olfactory neurons in both sexes. OrcoS289A mutants, unable to phosphorylate this position, have low intrinsic odorant sensitivity that is independent of altered expression or localization. A phosphomimetic allele, OrcoS289D, has enhanced odorant sensitivity compared with wild-type controls. To explore the functional ramifications of this phosphorylation in vivo, phospho-specific antiserum to OrcoS289 were generated, and it was shown that phosphorylation at this residue is dynamically regulated by odorant exposure with concomitant modulation of odorant sensitivity. OrcoS289 is phosphorylated in the sensitized state, and odorant exposure triggers dephosphorylation and desensitization without altering receptor localization. It was furthern show that dephosphorylation of OrcoS289 is triggered by neuronal activity, and not conformational changes in the receptor occurring upon ligand binding. Mutant flies unable to regulate Orco function through phosphorylation at S289 are defective for odor-guided behavior. These findings provide insight into the mechanisms underlying regulation of insect odorant receptors in vivo.
Trible, W., Olivos-Cisneros, L., McKenzie, S. K., Saragosti, J., Chang, N. C., Matthews, B. J., Oxley, P. R. and Kronauer, D. J. C. (2017). orco mutagenesis causes loss of antennal lobe glomeruli and impaired social behavior in ants. Cell 170(4): 727-735.e710. PubMed ID: 28802042
Summary:
Life inside ant colonies is orchestrated with diverse pheromones, but it is not clear how ants perceive these social signals. It has been proposed that pheromone perception in ants evolved via expansions in the numbers of odorant receptors (ORs) and antennal lobe glomeruli. This study generated the first mutant lines in the clonal raider ant, Ooceraea biroi, by disrupting orco, a gene required for the function of all ORs. orco mutants were found to exhibit severe deficiencies in social behavior and fitness, suggesting they are unable to perceive pheromones. Surprisingly, unlike in Drosophila melanogaster, orco mutant ants also lack most of the approximately 500 antennal lobe glomeruli found in wild-type ants. These results illustrate that ORs are essential for ant social organization and raise the possibility that, similar to mammals, receptor function is required for the development and/or maintenance of the highly complex olfactory processing areas in the ant brain.
Utashiro, N., Williams, C. R., Parrish, J. Z. and Emoto, K. (2018). Prior activity of olfactory receptor neurons is required for proper sensory processing and behavior in Drosophila larvae. Sci Rep 8(1): 8580. PubMed ID: 29872087
Summary:
Animal responses to their environment rely on activation of sensory neurons by external stimuli. In many sensory systems, however, neurons display basal activity prior to the external stimuli. This prior activity is thought to modulate neural functions, yet its impact on animal behavior remains elusive. This study reveals a potential role for prior activity in olfactory receptor neurons (ORNs) in shaping larval olfactory behavior. Prior activity in larval ORNs is shown to be mediated by the olfactory receptor complex (OR complex). Mutations of Orco, an odorant co-receptor required for OR complex function, cause reduced attractive behavior in response to optogenetic activation of ORNs. Calcium imaging reveals that Orco mutant ORNs fully respond to optogenetic stimulation but exhibit altered temporal patterns of neural responses. These findings together suggest a critical role for prior activity in information processing upon ORN activation in Drosophila larvae, which in turn contributes to olfactory behavior control.
Mysore, K., Li, P. and Duman-Scheel, M. (2018). Identification of Aedes aegypti cis-regulatory elements that promote gene expression in olfactory receptor neurons of distantly related dipteran insects. Parasit Vectors 11(1): 406. PubMed ID: 29996889
Summary:
Aedes aegypti is a vector mosquito that displays sexually dimorphic behaviors that contribute to pathogen transmission. Formaldehyde-assisted isolation of regulatory elements followed by sequencing (FAIRE-seq) has facilitated genome-wide discovery of A. aegypti cis-regulatory elements (CREs). The goal of this investigation was to identify FAIRE DNA elements that promote gene expression in the olfactory system. Eight A. aegypti CREs that promote gene expression in antennal olfactory receptor neurons (ORNs) were identified in a Drosophila melanogaster transgenic reporter screen. Four CREs identified in the screen were cloned upstream of GAL4 in a transgenic construct that is compatible with transformation of a variety of insect species. These constructs, which contained FAIRE DNA elements associated with the A. aegypti odorant coreceptor (orco), odorant receptor 1 (Or1), odorant receptor 8 (Or8) and fruitless (fru) genes, were used for transformation of A. aegypti. The CREs drove transgene expression in A. aegypti that corresponded to endogenous gene expression patterns of the orco, Or1, Or8 and fru genes in the mosquito antenna. These results provide further evidence that FAIRE-seq, which can be paired with D. melanogaster reporter screening to test FAIRE DNA element activity in select tissues, is a useful method for identification of mosquito cis-regulatory elements. These findings expand the genetic toolkit available for the study of Aedes neurobiology. Moreover, given that the CREs drive comparable olfactory neural expression in both A. aegypti and D. melanogaster, it is likely that they may function similarly in multiple dipteran insects, including other disease vector mosquito species.
BIOLOGICAL OVERVIEW

Fruit flies are attracted by a diversity of odors that signal the presence of food, potential mates, or attractive egg-laying sites. Most Drosophila olfactory neurons express two types of odorant receptor genes: Or83b, a broadly expressed receptor of unknown function, and one or more members of a family of 61 selectively expressed receptors. While the conventional odorant receptors are highly divergent, Or83b is remarkably conserved between insect species. Two models could account for Or83b function: it could interact with specific odor stimuli independent of conventional odorant receptors, or it could act in concert with these receptors to mediate responses to all odors. The results support the second model. Dendritic localization of conventional odorant receptors is abolished in Or83b mutants. Consistent with this cellular defect, the Or83b mutation disrupts behavioral and electrophysiological responses to many odorants (Larsson, 2004).

The olfactory system has evolved the capacity to recognize and discriminate an inordinate number of chemically distinct odors that signal the presence of food, predators, or mating partners. The initial steps in odor detection involve the binding of a volatile odor to odorant receptor (OR) proteins displayed on ciliated dendrites of specialized olfactory sensory neurons (OSNs) that are exposed to the external environment. The OR genes that mediate odor detection in Drosophila fruit flies are expressed in subpopulations of OSNs and are members of a rapidly diverging superfamily of insect chemosensory genes that encode receptors with no homology to nematode or vertebrate ORs. Genetic analysis coupled with electrophysiology has demonstrated that the characteristic odor response profile of a given OSN is governed by the selective expression of one or more members of the family of 61 OR genes in that neuron (Dobritsa, 2003; Elmore, 2003; Hallem, 2004a; Hallem, 2004b). One member of the OR gene family, Or83b, is strikingly different from the other OR genes. Unlike the conventional ORs, it has clear homologs in other insect species that share nearly 70% amino acid identity with Or83b. Or83b and its homologs in other insects are coexpressed with conventional ORs in a large proportion of OSNs (Vosshall, 1999: Vosshall, 2000; Elmore, 2003; Hill, 2002; Krieger, 2003; Pitts, 2004; Larsson, 2004 and references therein).

Based on these observations, two models could account for Or83b function in insect olfaction. Or83b could bind distinct ligands independent of the other OR genes coexpressed with it in a given OSN, or it could act in concert with conventional ORs to recognize a wide variety of odors (Larsson, 2004).

In the first model, Or83b might act in most OSNs to recognize and report the presence in the environment of an important odor signifying danger or a particularly rich food source. Insects have evolved a diversity of food preferences and inhabit many different ecological niches. However, the vast majority of insects have an important relationship to plants, either as food sources, sites for egg-laying, habitat for prey, or shelter. Green leaf volatiles such as E2-hexenal and linalool are produced by many different plants and elicit physiological and behavioral responses in insects as varied as moths, mosquitoes, and Drosophila. Therefore, Or83b might interact selectively with various plant volatiles and transmit the same information to insects of diverse taxonomy; i.e., that they are in the presence of plants. Alternatively, Or83b could interact with a single odorant that has different meanings to different insects. For instance, isoamyl acetate is a key component of rotting fruit and signifies food to Drosophila, while the same odorant is produced by honeybees as an alarm and aggregation pheromone. In both cases, the stimulus is of great importance to these insects, and dedicating a broadly expressed receptor like Or83b to detecting it might be adaptive for each insect. In either variation of this first model, animals lacking Or83b would show essentially normal responses to general odors detected by conventional ORs but would be nonresponsive to the putative important odors of special meaning (Larsson, 2004).

In the second model, Or83b would not function independently as a ligand binding OR but would play a more general role in concert with the conventional ORs with which it is coexpressed. Such alternative roles could include interacting with conventional ORs to produce a receptor complex competent for ligand binding, acting as a protein chaperone that directs ORs to the dendrite, the OSN, or a combination of all of these functions. In this scenario, severe deficits in olfactory function to a wide range of odors would be expected in animals lacking Or83b (Larsson, 2004).

Despite the remarkable progress in recent years in elucidating the molecular logic of olfaction in Drosophila, existing data do not explicitly rule out either model. Electrophysiological analysis of the basiconic class OSNs reveal a diversity of responses to odorants that included activation and inhibition of a given OSN by different odorants. OSNs were identified with specialist and generalist properties, and a number of identified neurons did not respond to any of the approximately 50 odorants tested. Available data on the response properties of the remaining OSNs in the antenna associated with trichoid and coeloconic sensilla are less complete. The hypothesized odorant of special importance that activates Or83b independent of the other ORs might lie outside of collection of odorants used in these studies or might emerge from analysis of the trichoid and coeloconic OSNs. Elegant genetic and functional analysis of Drosophila ORs produced the clear result that the response properties of an identified OSN require the expression of its cognate OR and that replacing this OR with a novel OR alters the response properties of the neuron in an OR-dependent manner. While these results clearly implicate the conventional OR in the response properties of the OSN, they do not rule out the possibility that these ORs function in concert with Or83b, which is coexpressed with all of these ORs (Elmore, 2003). They also do not rule out an independent function for Or83b. Or22a/b and Or43b mutant neurons were not found to respond to any of the odorants tested, but the relevant odorant might not have been part of the stimulus panel (Larsson, 2004).

To distinguish between these competing models for Or83b function, gene targeting was used to delete the Or83b gene, and cell biological, electrophysiological, and behavioral techniques were used to characterize the mutant phenotype. Clear support for the second model is found: in Or83b mutants, the normal localization of OR proteins in distal chemosensory dendrites is disrupted in both larval and adult olfactory systems. Or83b mutant larvae fail to chemotax to most odors tested, and adult flies show severe deficits in odor-evoked electrophysiology and behavior. These data therefore imply that olfactory function in response to a broad range of odorants in Drosophila requires expression of a conventional OR along with Or83b in most olfactory neurons. These findings have important implications for control of medically and economically relevant insect pests, because clear homologs of Or83b exist in malaria mosquitoes and a variety of important agricultural pests (Krieger, 2003; Pitts, 2004). These data suggest a strategy in which olfactory host-seeking behavior of pest insects could be disrupted by small molecule inhibitors of Or83b homologs (Larsson, 2004).

This study shows, by cellular, physiological, and behavioral analysis, that Or83b is essential for olfaction in Drosophila. Or83b is an atypical member of the OR gene family because it is highly conserved across insect species and is expressed in a large number of OSNs with different odor specificities (Dobritsa, 2003; Elmore 2003; Hallem, 2004a). This receptor is selectively expressed only in OSNs throughout all four stages of Drosophila development, and no expression is detected in gustatory neurons or any other cell type. Fly Or83b is expressed in all OSNs of the larval dorsal organ and adult maxillary palp and in a large subset of adult antennal neurons. The onset of Or83b expression in both larval and adult olfactory systems is late, effectively ruling out any developmental role for this protein in patterning axonal connections of these neurons (Larsson, 2004).

An Or83b mutant was constructed by 'ends-out' gene targeting and shown to be null for both mRNA and protein expression. Conventional OR proteins fail to accumulate in both adult and larval OSN dendrites in Or83b mutants and are restricted to the cell body. This suggests a role for Or83b in regulating the proper subcellular localization of the conventional ORs. The OR localization defect was shown to be specific to Or83b and not a secondary effect of a sick neuron. The distribution of a general membrane marker in larvae and the futsch microtubule marker is unaffected in the mutant, as is the stability of Or22a-expressing axonal connections to the DM2 glomerulus in the antennal lobe. Electrical silencing of wild-type neurons does not produce the same OR localization defect as the Or83b mutation (Larsson, 2004).

Or83b mutant antennae show no odor-evoked potentials to a panel of eight odorants that elicit robust responses in wild-type antenna. Mutant ab1A, B, and D neurons fail to respond to their cognate stimuli and show little or no spontaneous electrical activity. In contrast, the carbon dioxide-sensitive ab1C neuron is normal in the Or83b mutant. Finally, both larval and adult Or83b mutants have severe deficits in odor-evoked behavioral responses. Taken together, these data support a model in which Or83b acts in concert with conventional ORs to respond to many different odorants and argue against an independent function for Or83b in recognizing a particular odorant (Larsson, 2004).

The number of ORs expressed in a given OSN is an important determinant of the coding logic of the olfactory system. In the nematode, C. elegans, 16 pairs of chemosensory neurons express an estimated 500 different chemosensory receptor genes. Of necessity, each chemosensory neuron expresses a large number of receptors, but each receptor functions independently within a neuron to recognize ligands that activate the chemosensory neuron to elicit either attractive or aversive behavioral responses according to behavioral output, which is regulated by the activation of the sensory neuron: the animal retains great discriminatory power in the face of a severely constricted number of sensory neurons and no central chemosensory processing circuits. In contrast, each mammalian olfactory neuron expresses a single odorant receptor gene that bestows upon that neuron a restricted receptive range for odors. In part because the odorant receptor itself is an important determinant in glomerular target selection, mammalian olfactory neurons have evolved elaborate regulatory mechanisms to suppress the expression of more than one functional odorant receptor gene per neuron. Unlike the situation in C. elegans, each sensory neuron contributes information about a small fraction of the odor universe, and significant olfactory processing must occur in higher order olfactory cortical regions to decode the salience of the odor stimulus and produce an appropriate behavioral response (Larsson, 2004).

Initial analysis of the Drosophila OR gene family suggested that the fly olfactory system was organized according to the vertebrate one receptor:one neuron:one glomerulus model. OR genes were found in nonoverlapping subpopulations of OSNs, and all OSNs expressing a given OR were found to converge upon a distinct and dedicated antennal lobe glomerulus. However, further analysis has somewhat complicated this initial view. Or22a and Or22b are coexpressed in the ab3a neuron, that Or22a functions independently of Or22b, and that Or22b does not discernibly contribute to the odor code of this neuron. This same neuron expresses Or83b in addition to these two conventional odorant receptors. If each of these three ORs hypothetically interacts with distinct ligands, this would substantially alter the view of how the fly olfactory system is organized. In such a multireceptor OSN model diverse stimuli would activate the same neuron, but all would lead to activation of the DM2 glomerulus to which the neuron projects. The animal would thus have no means to determine which of the three receptors was activated, resulting in a possible loss of odor discrimination. The data strongly argue against an independent requirement of a single receptor protein for the function for Or83b in odor detection and instead suggest that it acts as an essential cofactor for localizing conventional ORs in chemosensory dendrites. Therefore, the fly is likely to retain an organizational logic similar to that employed in vertebrates, despite expressing more than one OR in each olfactory neuron (Larsson, 2004).

This study demonstrates a genetic requirement for Or83b in dendritic localization of conventional ORs in vivo and would be consistent with a model in which Or83b interacts with the conventional odorant receptors to form a heteromeric receptor complex. Conclusive evidence for direct association between Or83b and conventional ORs, as well as more detailed biochemical insights into the function of Or83b, awaits the development of an expression system that can be used to examine the interactions of biologically active Drosophila ORs (Larsson, 2004).

Functional interaction between chemosensory receptors has been described in mammalian taste, where the assembly of different heterodimeric receptors determines whether a neuron responds to sweet or amino acid stimuli: the T1R2/3 heterodimer encodes a sweet taste receptor, while T1R1/2 encodes the umami or amino acid receptor. Homodimeric T1R3 receptors detect only high concentrations of natural sweeteners such as sucrose. Therefore, in the mouse three different classes of gustatory receptor cells express T1R3 but the functional specificity of the cell is determined by whether a given cell expresses T1R2 (regular sweet), T1R1 (umami), or neither (low-affinity sweet) (Larsson, 2004).

Broad expression of atypical chemosensory receptor genes, such as described in this study for Or83b, has also been seen. In the rodent vomeronasal organ, members of the V2R2 pheromone receptor subfamily are broadly expressed along with other more selectively expressed receptors (see the PDF file Molecular Detection of Pheromone Signals in Mammals: From Genes To Behaviour), but biochemical evidence that they interact functionally is lacking. Amino acid detection in fish relies in part on a broadly expressed receptor of the same structural class as the T1R taste receptors and V2R pheromone receptors, but it is not known whether this 5.24 receptor functions in concert with other fish receptors with which it is coexpressed (Larsson, 2004).

The data on Or83b point to a unique and unprecedented requirement of a single receptor protein for the function for functionality of an extremely diverse family of receptors. These results are unexpected because the Drosophila odorant receptor genes are unrelated to other receptor superfamilies that have been shown to heterodimerize through conserved protein-protein interaction domains. Further, this receptor family is undergoing rapid evolutionary change with virtually no direct homologs recognized between the fly and mosquito genomes. In contrast, Or83b is extremely conserved and has clear homologs in distant insect species. If these homologs are performing the same function in these divergent insect species, it would follow that Or83b must recognize a conserved feature of all OR proteins with which it is expressed, either directly by heterodimerizing with the ORs or through the action of accessory proteins (Larsson, 2004).

The investment of Drosophila in a single protein for odor detection suggests that Or83b performs a key function in all insects that cannot be diversified or made redundant. The alternative to maintaining strong selection pressure on Or83b would be to require parallel selection on a diversity of other genes that each interact with a partner OR protein. Such a coevolution of two large gene families is suggested by the work on the M10 MHC family that associates with a family of V2R receptors in the vomeronasal organ (Ishii, 2003; Loconto, 2003). There may be barriers to the generation of large numbers of new genes in Drosophila, which maintains a compact genome with few pseudogenes (Larsson, 2004).

Insects are the primary vectors for the infectious diseases malaria, dengue fever, yellow fever, and West Nile encephalitis, and they locate human hosts largely through their exquisitely sensitive olfactory systems. Host-seeking behavior is thought to require a number of different sensory stimuli to provide the maximum likelihood that the human host is near. For instance, mosquitoes are attracted to general odors along with carbon dioxide and body heat. The finding that the Or83b mutation disrupts Drosophila behavioral responses to many odors suggests a potential chemical strategy to disrupt the function of homologous genes in pest insects. Small molecule inhibitors that mimic the effects of this mutation may blunt or eliminate olfactory responses in pest insects, ultimately controlling the damaging olfactory-mediated behaviors that result in the spread of disease (Larsson, 2004).

Insect olfactory receptors are heteromeric ligand-gated ion channels

In insects, each olfactory sensory neuron expresses between one and three ligand-binding members of the olfactory receptor (OR) gene family, along with the highly conserved and broadly expressed Or83b co-receptor. The functional insect OR consists of a heteromeric complex of unknown stoichiometry but comprising at least one variable odorant-binding subunit and one constant Or83b family subunit. Insect ORs lack homology to G-protein-coupled chemosensory receptors in vertebrates and possess a distinct seven-transmembrane topology with the amino terminus located intracellularly. This study provides evidence that heteromeric insect ORs comprise a new class of ligand-activated non-selective cation channels. Heterologous cells expressing silkmoth, fruitfly or mosquito heteromeric OR complexes show extracellular Ca2+ influx and cation-non-selective ion conductance on stimulation with odorant. Odour-evoked OR currents are independent of known G-protein-coupled second messenger pathways. The fast response kinetics and OR-subunit-dependent K+ ion selectivity of the insect OR complex support the hypothesis that the complex between OR and Or83b itself confers channel activity. Direct evidence for odorant-gated channels was obtained by outside-out patch-clamp recording of Xenopus oocyte and HEK293T cell membranes expressing insect OR complexes. The ligand-gated ion channel formed by an insect OR complex seems to be the basis for a unique strategy that insects have acquired to respond to the olfactory environment (Sato, 2008).

Taken together, these data provide compelling evidence that a heteromeric complex of a conventional insect OR and the highly conserved Or83b family co-receptor has the characteristics of a cation non-selective ion channel directly gated by odour or pheromone ligands. It is concluded that G-protein-mediated signalling is negligible in producing the current elicited by odour activation of insect OR heteromultimers. These findings provide insight into long-argued insect olfactory transduction mechanisms and may explain the lack of clear consensus on the role of second messengers in this process. The insect ORs share no homology with any previously described ion channel and do not contain any known ion selectivity filter motifs. Insect OR activity is not inhibited by Gd3+, a lanthanide that is a broad-spectrum ion channel inhibitor. Therefore, although the ionic permeability reported in this study for Na+, K+ and Ca2+ would be consistent with the properties of non-selective cation channels, a molecular basis for this novel ionotropic activity remains to be elucidated. The spontaneous activity of the OR complex found in this study seems to account for previous observations that olfactory sensory neurons exhibit bipolar electrical activity and become electrically negative on the deletion of Or83b in vivo. Given that there are 62 and 79 potential ligand-binding OR subunits in Drosophila and Anopheles, respectively, the insect ORs may represent the largest single family of ion-channel-like proteins in any organism. This work also raises the intriguing possibility that the insect gustatory system, which senses bitter and sweet tastants as well as carbon dioxide, shares this ionotropic coupling mechanism with the insect ORs. In fact, an ionotropic sugar-gated channel in fleshfly taste cells has previously been reported. This finding offers the caveat that other orphan receptors classified as G-protein-coupled receptors merely because of their putative seven-transmembrane topology may instead possess ligand-gated channel activities, as has been shown previously for light-activated channelrhodopsin. This work has important implications for worldwide efforts to identify specific inhibitors for the insect ORs, which may prove useful in controlling host-seeking behaviours of disease-vector insects such as mosquitoes (Sato, 2008).

From worm to man, many odorant signals are perceived by the binding of volatile ligands to odorant receptors1 that belong to the G-protein-coupled receptor (GPCR) family. They couple to heterotrimeric G-proteins, most of which induce cAMP production. This second messenger then activates cyclic-nucleotide-gated ion channels to depolarize the olfactory receptor neuron, thus providing a signal for further neuronal processing. Recent findings, however, have challenged this concept of odorant signal transduction in insects, because their odorant receptors, which lack any sequence similarity to other GPCRs, are composed of conventional odorant receptors (for example, Or22a), dimerized with a ubiquitously expressed chaperone protein, such as Or83b in Drosophila. Or83b has a structure akin to GPCRs, but has an inverted orientation in the plasma membrane. However, G proteins are expressed in insect olfactory receptor neurons, and olfactory perception is modified by mutations affecting the cAMP transduction pathway. This study shows that application of odorants to mammalian cells co-expressing Or22a and Or83b results in non-selective cation currents activated by means of an ionotropic and a metabotropic pathway, and a subsequent increase in the intracellular Ca2+ concentration. Expression of Or83b alone leads to functional ion channels not directly responding to odorants, but being directly activated by intracellular cAMP or cGMP. Insect odorant receptors thus form ligand-gated channels as well as complexes of odorant-sensing units and cyclic-nucleotide-activated non-selective cation channels. Thereby, they provide rapid and transient as well as sensitive and prolonged odorant signalling (Wicher, 2008).

Insect odorant response sensitivity is tuned by metabotropically autoregulated olfactory receptors

Insects possess one of the most exquisitely sensitive olfactory systems in the animal kingdom, consisting of three different types of chemosensory receptors: ionotropic glutamate-like receptors (IRs), gustatory receptors (GRs) and odorant receptors (ORs). Both insect ORs and IRs are ligand-gated ion channels, but ORs possess a unique configuration composed of an odorant-specific protein OrX and a ubiquitous coreceptor (Orco). In addition, these two ionotropic receptors confer different tuning properties for the neurons in which they are expressed. Unlike IRs, neurons expressing ORs are more sensitive and can also be sensitized by sub-threshold concentrations of stimuli. What is the mechanistic basis for these differences in tuning? This study shows that intrinsic regulation of Orco enhances neuronal response to odorants and sensitizes the ORs. It was also demonstrated that inhibition of metabotropic regulation prevents receptor sensitization. These results indicate that Orco-mediated regulation of OR sensitivity provides tunable ionotropic receptors capable of detecting odors over a wider range of concentrations, providing broadened sensitivity over IRs themselves (Getahun, 2013).

The independent evolution of these two different ionotropic receptor families (ORs/GRs and IRs) has become a great topic of speculation for the field. Why do these multiple families persist among all higher insect orders? And why do they possess such radically different molecular conformations? Initially, it was suggested that these multiple families expand the affinity of the olfactory palette to different chemical classes. However, a recent study also revealed that olfactory sensory neurons (OSNs) expressing ORs, GRs, or IRs exhibit intrinsic differences in temporal kinetics to brief or intermittent stimuli (Getahun, 2012). Specifically, OR-expressing neurons respond faster and with higher sensitivity to brief stimulation, while IR-expressing neurons do not adapt to long stimulations. This implies that OR-expressing neurons are more accurate at detecting the low-concentration, punctate plume packets received at long distances from the odor source, while IR-expressing neurons can better track the high-concentration, long lasting stimulation received when on or near the source. This diversity offers both broader ligand specificity and expanded spatiotemporal dynamics with which to parse the odor world, and is particularly important for insects challenged by the high-speed performance of flight. Interestingly, the purported evolution of ORs corresponds well to the evolution of flight during the Carboniferous Era (Getahun, 2013).

Although both insect ORs and IRs operate as ionotropic receptors, their tuning properties differ fundamentally. While prolonged stimulation leads to adaptation of ORs, there is no adaptation of IRs. On the other hand, ORs but not IRs expand their dynamic range through intrinsic sensitization. This difference in sensitization is apparent even between ORs and IRs expressed in co-localized sensilla. Thus, sensitization must result from intrinsic, rather than extrinsic neuronal properties that are unique to ORs. The most parsimonious explanation for the mechanistic differences between these families, is the use of intracellular signalling to modulate OR activity. Given the previous in vivo evidence for a role of metabotropic signalling in OR function, this study first pursued the metabotropic regulation of Orco in mediating OR activity (Getahun, 2013).

OR sensitization could be mimicked by manipulations enhancing cAMP production or PKC activity and depressed by inhibition of cAMP production or PLC/PKC activity. These intracellular signalling systems not only influence the OR sensitivity at weak odor stimuli, they also modulate the OR response for stronger stimuli. In detail, microinjection of cAMP or adenylyl cyclase activators into sensilla increased the odorant response and shifted the dose-response curve toward lower odorant concentrations. A previous study has revealed that Orco sensitivity to cAMP is regulated by protein kinase C (PKC)-dependent phosphorylation (Sargsyan, 2011). Inhibition of PLC or PKC also inhibits any effect of cAMP, indicating that the enhanced sensitivity caused by cAMP is regulated by Orco activity. The metabotropic regulation of Orco also lead to sensitization of the OSN to repeated subthreshold odor responses, which is abolished by adenylyl cyclase inhibition. Furthermore, the sensitization of the odor response was blocked in mutant flies with impaired Orco phosphorylation (Orco mut) further indicating that metabotropic regulation of Orco activity is required for the enhanced odorant response. It cannot be excluded that cAMP and PKC activation may regulate OR sensitivity to odors via other mechanisms, such as through modulation of membrane traffick. Nevertheless, the lack of response modulation following injection of forskolin into PKC flies, indicates that the metabotropically-enhanced odor sensitivity is intrinsic to the OR complex and does not result from extrinsic cellular processes (Getahun, 2013).

The results thus suggest that intracellular signalling, and in particular metabotropic regulation of Orco, plays a vital role in conferring the mechanistic differences between ORs and IRs. Although the mechanistic basis of intracellular signalling in these OSNs cannot yet be machanistically confirmed, it is concluded that modulations that activate Orco when heterologously expressed enhance the odor sensitivity of ORs in vivo and, vice versa, modulations that inhibit Orco reduce OR sensitivity. It must also be kept in mind that the ORs are Ca2+-permeable, constitutively active ion channels, the background activity of which is also able to activate enzymatic activity. Future studies should characterize the composition of the respective signalling subsystems, e.g. those involved in sensitizing receptors vs. those involved in terminating the odorant response (Getahun, 2013).

The evolution of a highly sensitive and adaptable olfactory system is believed to be a key factor allowing insects to radiate into more or less every environment on earth. Given the importance of OSN dynamics in tracking turbulent odor plumes, olfactory sensitization via Orco regulation can enhance an insect's ability to accurately detect and respond to intermittent, low concentration stimuli. Insect ORs are thought to have evolved from ionotropic gustatory receptors, which detect millimolar ligand concentrations. The results imply that the special heterodimeric design of ORs has likely evolved to quickly detect and respond to volatile compounds at very low concentrations, such as those encountered by flying insects. Regardless of the source of this difference, it is clear that the OR expansion of ionotropic receptors offers the insect olfactory system both broadened ligand affinity as well as expanded spatiotemporal dynamics with which to navigate the olfactory world (Getahun, 2013).


REGULATION

Chemotaxis behavior mediated by single larval olfactory neurons in Drosophila

Odorant receptors (ORs) are thought to act in a combinatorial fashion, in which odor identity is encoded by the activation of a subset of ORs and the olfactory sensory neurons (OSNs) that express them. The extent to which a single OR contributes to chemotaxis behavior is not known. This question was investigated in Drosophila larvae, which represent a powerful genetic system to analyze the contribution of individual OSNs to odor coding. Twenty-five larval OR genes expressed in 21 OSNs were identified and genetic tools were generate that allow engineering of larvae missing a single OSN or having only a single or a pair of functional OSNs. Ablation of single OSNs disrupts chemotaxis behavior to a small subset of the odors tested. Larvae with only a single functional OSN are able to chemotax robustly, demonstrating that chemotaxis is possible in the absence of the remaining elements of the combinatorial code. Behavioral evidence is provided that an OSN not sufficient to support chemotaxis behavior alone can act in a combinatorial fashion to enhance chemotaxis along with a second OSN. It is concluded that there is extensive functional redundancy in the olfactory system, such that a given OSN is necessary and sufficient for the perception of only a subset of odors. This study is the first behavioral demonstration that formation of olfactory percepts involves the combinatorial integration of information transmitted by multiple ORs (Fishilevich, 2005).

The 'nose' of the Drosophila larva resides in a pair of dorsal organs at the anterior tip of the animal, each containing 21 OSNs. Previous studies showed that up to 23 of the 61 Drosophila ORs are expressed in larvae by PCR and transgenic analysis. RNA in situ hybridization was performed to provide direct evidence that OR genes are expressed in larval OSNs. Or83b, which is necessary for the proper localization and function of conventional ORs, is broadly expressed throughout the dorsal-organ ganglion. Twenty-four of the 30 ORs tested in this study are expressed in a single larval neuron in the dorsal organ. The expression of Or10a, Or43b, or Or49a mRNA or OR43b protein was not detected, although RT-PCR analysis detects these transcripts in larvae. Or92a and Or98b are also not detected by RNA in situ hybridization. Most larval OSNs express a single OR along with Or83b, but two OSNs coexpress a pair of ORs along with Or83b: Or33b/Or47a and Or94a/Or94b. Such OR coexpression has also been documented in the adult olfactory system (Fishilevich, 2005).

In parallel with the RNA in situ hybridization analysis, a collection of 42 different OR-Gal4 transgenes were examined that drive the expression of Gal4 under the control of OR promoter elements. To visualize gene expression in the dorsal organ, individual OR-Gal4 lines were crossed to UAS-GFP, encoding cytoplasmic green fluorescent protein (GFP) and the olfactory-neuron marker Or83b-Myc. Or83b-Gal4 labels all 21 larval OSNs. Per dorsal organ, 19 of the remaining 41 OR-Gal4 transgenes label a single larval OSN that is also positive for Or83b-Myc. Although Or49a mRNA was not detected in larvae, Or49a-Gal4 labels one dorsal-organ OSN along with a single terminal-organ gustatory neuron. Gustatory receptor (GR) genes are expressed in both olfactory and gustatory organs of the adult fly. GR-Gal4 transgenes are expressed only in the gustatory terminal organ or in nonolfactory dorsal-organ neurons that do not express Or83b-Myc. A total of 25 Drosophila ORs expressed in the larval dorsal organ were identified and direct evidence is provided that 24 of these OR mRNAs are expressed in situ. Of these, 14 are expressed only at the larval stage, whereas 11 are utilized by both larval and adult olfactory systems (Fishilevich, 2005).

Larval OSNs project axons to the larval antennal lobe of the brain. Patterns of axonal projections to the larval antennal lobe were examined in larvae carrying each of 20 larval OR-Gal4 transgenes along with UAS-GFP or UAS-CD8-GFP. Each OR-Gal4 line reveals a single labeled axonal arbor that terminates in an antennal-lobe glomerulus whose position is conserved between animals (Fishilevich, 2005).

The availability of genetic tools that uniquely label 19 of the 21 larval OSNs allows manipulation of the odor code by deconstructing the peripheral olfactory input and examining effects on behavioral output. Toward this end, a chemotaxis assay was establised of sufficient sensitivity to quantify differences in odor-evoked behavior. Chemotaxis of wild-type larvae was measured in response to 53 synthetic monomolecular odorants and three natural Drosophila attractants. The assay involves single-animal analysis in which the position of individual chemotaxing larvae is tracked over the course of a 5 min experiment (Fishilevich, 2005).

This assay was used to screen larval chemotaxis to a panel of 53 synthetic odors and quantified the median distance to odor for Or83b−/− and Or83b+/+ larvae. Forty of the 53 odors are naturally present in fruit, and of these 40, 13 are known to elicit behavioral and electrophysiological responses in Drosophila. Anosmic Or83b−/− larvae do not respond to any odors, but wild-type (yw) larvae respond to many odors with strong chemotaxis (Fishilevich, 2005).

It was next asked how sensitive larvae are to odors by performing chemotaxis experiments at various odor concentrations. The responses to 1-hexanol are weak and not statistically different from anosmic controls for low dilutions, whereas responses increase steeply between 0.02 μl and 0.2 μl doses and appear to reach a plateau for higher concentrations. No evidence was found that higher concentrations elicit repulsion. Response thresholds to heptanal and isoamyl acetate are one and two log orders, respectively, below that of 1-hexanol (Fishilevich, 2005).

To test whether the weak responses observed for some odors at 2 μl could be explained by high detection thresholds, seven of these odors were further tested with 20 μl. Under these conditions, 1-butanol and 2,3-butanediol elicit chemotaxis, whereas the remaining five odors do not. Thus the 2 μl stimulus dose elicits robust chemotaxis across a large group of different odors, in accord with previous behavioral studies (Fishilevich, 2005).

Upon loading of an odorant stimulus in the closed-dish assay, the spatial distribution and average airborne concentration of this odor in the dish will be partly determined by the odor's vapor pressure. Vapor pressure is thus likely to affect the behavioral response observed for a particular odorant stimulus. In addition to this factor, it is anticipated that the olfactory system of the larva may be differentially tuned to different stimuli. In the initial phases of this study, no clear correlation was found between the vapor pressure of a given odor and its corresponding behavioral efficacy. It was therefore decided to avoid any normalization of stimulus concentration and used the same quantity of odor (2 μl) for all 53 stimuli tested (Fishilevich, 2005).

Whether chemotaxis elicited by single odors is comparable to that obtained with natural stimuli was examined. Chemotaxis was measured in the same assay to banana mush, balsamic vinegar, and yeast paste at different concentrations. It was found that attraction elicited by single synthetic odors is qualitatively similar to that obtained with natural odor blends and that the same steep threshold and stable plateau properties are seen for both stimulus types (Fishilevich, 2005).

The relative contribution of any given OSN to the formation of an odor percept was examined. Diphtheria toxin (DTI), an attenuated version of the cell-autonomous protein-translation inhibitor diphtheria toxin, was used to ablate identified OSNs selectively. Most but not all larval OSNs are ablated by the expression of DTI along with GFP under control of the Or83b-Gal4 driver in all 21 larval OSNs. In Or83b-ablated animals, GFP expression is not detected, and sensory dendrites are severely atrophied but not completely absent. In Or49a-ablated animals, the Or49a-GFP marker is not visible, and expression of other ORs is not perturbed (Fishilevich, 2005).

Chemotaxis of animals with single neurons ablated (Or1a, Or42a, or Or49a) was measured with a panel of 20 odors and compared to results obtained with the Or83b-ablation. Or83b-ablated larvae fail to respond to 17/20 odors. If a single false discovery (FD) is allowed for, Or83b-ablated animals fail to respond to 19/20 odors. Or1a-ablated and Or49a-ablated animals each show reduced chemotaxis to a single different odor, (E)-2-hexenal and 1-hexanol, respectively, but show normal chemotaxis to the other 19 odors. In contrast, ablation of the Or42a OSN causes decreases in chemotaxis to four of 20 odors. If FD = 1 is allowed, Or1a-ablated animals are impaired in responses to three of 20 and Or42-ablated animals to five of 20 odors (Fishilevich, 2005).

It was next asked which OSNs are sufficient to produce chemotaxis to a given odor by constructing animals with only one or combinatorials of two functional OSNs. This was achieved by exploiting the Or83b mutation, which prevents OR trafficking to the sensory dendrite. Or83b function was restored in individual OSNs by crossing animals with specific OrX-Gal4 drivers to UAS-Or83b animals, allowing assessment of the contribution of single neurons to odor-evoked behavior in the OrX-functional progeny (Fishilevich, 2005).

Only a single OR83b-expressing neuron is seen in Or42a-functional, Or49a-functional, and Or1a-functional animals, whereas two OR83b-positive neurons are visible in Or1a-/Or42a-functional and Or1a-/Or49a-functional animals. The remaining OSNs are present but unlabeled in these animals because the Or83b mutation eliminates OR83b protein expression. No evidence was found that the glomerular map is distorted by the activation of a single OSN in a background of nonfunctional neurons as evidenced by the normal position and volume of the Or1a glomerulus in Or1a-functional and Or83b mutant larvae (Fishilevich, 2005).

These animals along with genetically matched control animals were screened for chemotaxis to 53 odors by using the same behavioral assay and nonparametric statistical analysis employed for the ablation experiments. Consistent with the strong Or42a-ablated phenotypes, Or42a-functional animals respond to 22 odors compared to 36 odors in Or83b+/+ controls possessing 21 functional OSNs. Or42a-functional animals respond to three of four odors to which Or42a-ablated animals are anosmic. The broad behavioral response profile observed for Or42a-functional larvae is in agreement with the broad ligand specificity of this OR as defined by electrophysiological experiments (Fishilevich, 2005).

In contrast to the broad odor response profile of Or42a-functional larvae, Or1a- and Or49a-functional animals do not show significant chemotaxis to any of the 53 odors tested, consistent with the weak phenotype of ablating either the Or49a-expressing or Or1a-expressing neuron. These behavioral results are in accord with the ligand profiling of Or49a, which does not show strong electrophysiological responses to any of 27 odors tested (Fishilevich, 2005).

Although Or1a- and Or49-functional larvae do not chemotax to any odors tested, it was asked whether these neurons contribute to chemotaxis in concert with the Or42a neuron. Chemotaxis performance of larvae with two functional neurons was compared to data from animals with only a single functional neuron. Larvae with two functional neurons respond to a somewhat different subset of odors than animals having either single functional neuron alone (Fishilevich, 2005).

To examine the existence of interactions between these neurons and identify cases of combinatorial enhancement, a linear regression model was developed to compare chemotaxis data across genetically matched controls for larvae with one or two functional OSNs. The model was designed to identify potential cases where single-neuron chemotaxis behavior differs from two-neuron behavior. The linear model suggests six cases of potential positive cooperativity between Or1a and Or42a chemotaxis that merited further experimental investigation. Additional chemotaxis experiments were carried out with four odors (1-pentanol, 2-pentanol, 2-hexanol, and 3-octanone) at three concentrations. 1-pentanol shows significantly stronger chemotaxis in Or1a/Or42a-functional animals than Or42a-functional or Or1a-functional animals at all three concentrations. A qualitative view of this behavioral enhancement is seen in the sector-plot distributions comparing the anosmia of Or83b mutants to the progressive increase in chemotaxis to 1-pentanol of Or1a-functional or Or42a-functional compared to Or1a/Or42-functional. The Or1a/Or42-functional animals spend comparatively more time in the sector containing the odor than animals having either single functional neuron alone. For the other three odors, this cooperative effect is significant at a single odor concentration (Fishilevich, 2005).

This study has used behavioral analysis to measure the contribution of individual neurons to the odor code and provide a missing link between the understanding of the molecular biology of ORs, the neurophysiological properties of the olfactory network, and complex odor-evoked behaviors. The goal was to approach the question of how the combinatorial activation of ORs encodes odor stimuli and elicits olfactory behavior. The results suggest that there is a high level of redundancy in the larval olfactory system, such that ablating a single neuron has minimal effects on odor detection. Among these olfactory inputs, the Or42a neuron plays a more important role in odor detection than the Or1a or Or49a neuron. Animals engineered to have the Or42a neuron functional are able to chemotax to multiple odors. The addition of a second OSN to such animals results in enhanced chemotaxis for several odors. Whereas Or1a-functional animals show no significant responses to any odor tested, it was observed that responses of Or1a/Or42a-functional animals to four odors are enhanced relative to Or42a-functional animals. This suggests that although olfactory input contributed by the Or1a-expressing OSN is not sufficient alone to elicit robust chemotaxis, it enhances the perception of odors in conjunction with the information transmitted by the Or42a-expressing OSN. (Fishilevich, 2005).

Behavior is the ultimate output of a sensory system that integrates all aspects of external-information processing. These experiments demonstrate the feasibility and value of integrating behavioral analysis into the study of odor coding. It is proposed that the simple olfactory system of Drosophila larvae will be an invaluable model in any attempt to correlate the cellular basis of the odor code with its behaviorally relevant output (Fishilevich, 2005).

Drosophila is a holometabolous insect that undergoes dramatic changes in lifestyle from the larval to adult stage. In a sense, these animals can be considered to occupy completely separate ecological niches. Larvae maintain constant contact with food until pupation, whereas adults are flying insects that use their sense of smell to identify suitable food sources and appropriate sites for egg-laying. In essence, larvae are specialized for feeding and growth, whereas adults are devoted to breeding and dispersal. To what extent have these two life stages of the same species evolved a different chemosensory system? This study shows 14 of 25 larval OR genes are stage specific and not used again by the adult animal. All larval OSNs are histolyzed in metamorphosis and replaced in the adult by newly differentiated antennal and maxillary palp OSNs. Perhaps this developmental changeover has led to largely separate OR genes with transcriptional regulatory regions specific for either larval or adult olfactory organs. Alternatively, the segregation of larval- and adult-expressed ORs could be functional and relate to the different ecological niches that these life stages occupy: larvae may cope with much higher odor concentrations because of their direct contact with food (Fishilevich, 2005).

Odor processing occurs at various levels in the nervous system, from peripheral sensory neurons to primary processing centers, such as the olfactory bulb in vertebrates and the antennal lobe in insects, and further to higher brain centers of the olfactory cortex in vertebrates and mushroom body and lateral horn in insects. How the combinatorial code established by the ORs at the periphery is transmitted through this olfactory circuitry to produce the perception of an odor in any species is unknown. The data support the notion that peripheral sensory neurons constitute information channels that are not independent but subject to interactions in the olfactory circuit. Otherwise, one would expect that the behavioral response profile observed for the Or1a/Or42a-functional genotype be given by the union of the best performances of the single Or1a- and Or42a-functional genotypes. Where and how the information is processed remains unclear, but part of this transformation may occur in the antennal lobe (Fishilevich, 2005).

A number of conclusions about odor coding in the Drosophila larva can be drawn from this work. There appears to be no clear structural relationship between the odors that elicit chemotaxis mediated by a given OSN, as has been previously shown in an analysis of the ligand response properties of ORs in the adult fly. The Or42a-expressing neuron differs from other neurons studied here in the large number of odors that attract animals having only this neuron active. Interestingly, the behavioral response profile of the Or42a-functional genotype indicates that an OR may not need to be strongly activated by a given odor to allow for chemotaxis toward the odor source. This point is best illustrated by 3-octanol and anisole, which both elicit strong chemotaxis in Or42a-functional animals whereas they seem to induce relatively weak electrophysiological activity (Fishilevich, 2005).

Finally, the behavioral receptive field of animals having combinatorials of functional neurons cannot be predicted from a simple model where the responses of animals having either single OSN functional are added. The chemotaxis results reported in this study highlight the existence of strong nonlinearities in the processing of olfactory information in such a way that in the arithmetic of sensory coding, the whole is greater than what the parts can produce independently. Such a scheme would be consistent with the extraordinary needs of the olfactory system to detect numbers of odors that greatly exceed the number of OR genes in any given animal. The functional redundancy observed here could buffer the olfactory system against mutations and allow animals to adapt to changing or new odor environments (Fishilevich, 2005).

The genetic tools presented in this study should permit a systematic analysis of the peripheral and central components that generate an odor response in the Drosophila larva. A number of key unanswered questions remain for future studies. Electrophysiological or optical imaging tools must be used to analyze the neuronal correlates of the observed behavior. Greater understanding of the second- and third-order neurons that communicate information from the antennal lobe to eventual motor output is needed. This study has been restricted to simple chemotaxis assays, and no attempt has been made to query larvae for their powers of odor discrimination. Animals missing a single OSN may chemotax normally but experience olfactory-perception not uncovered in these chemotaxis assays. By coupling associative learning of odors in intact animals followed by generalization tests in the same animals that conditionally lack a single OSN, it should be possible to determine whether odor salience is altered in larvae missing a single OSN. Finally, it will be important to determine whether the phenomena reported in this study can be considered general olfactory-coding principles that also apply to more complex animals (Fishilevich, 2005).

Protein Interactions

Odorant receptor heterodimerization in the olfactory system of Drosophila

Despite increasing knowledge about dimerization of G-protein-coupled receptors, nothing is known about dimerization in the largest subfamily, odorant receptors. Using a combination of biochemical and electrophysiological approaches, this study demonstrates that odorant receptors can dimerize. DOR83b, an odorant receptor that is ubiquitously expressed in olfactory neurons from Drosophila and highly conserved among insect species, forms heterodimeric complexes with other odorant-receptor proteins, which strongly increases their functionality (Neuhaus, 2005).

Generally, a given odorant-receptor gene is expressed in only a small fraction of olfactory receptor neurons (ORNs), and each ORN expresses a very small number of odorant-receptor genes. In insects, however, one highly conserved member of the odorant-receptor gene family is expressed in nearly all ORNs in addition to the conventional odorant receptor or receptors. The encoded proteins DOR83b in Drosophila, AgOr7 in Anopheles gambiae, HvirR2 in Heliothis virescens and AmelR2 in Apis mellifera have between 64% and 88% amino acid-sequence identity, a level not observed for any other insect odorant-receptor gene. The function of this ubiquitously expressed protein is still unknown. Antennal neurons in Drosophila that express only DOR83b do not respond to any of a large panel of odors, so it is unlikely that the receptor itself has ligand-binding properties. Alternatively, DOR83b could act as a subunit in heterodimeric receptor complexes, either modulating the binding specificity of the coexpressed receptor or regulating the assembly of functional receptor transduction complexes, both of which are known functions of G-protein-coupled receptors. Heterodimerization might alternatively be necessary for the folding or membrane targeting of conventional odorant-receptor proteins. Using a combination of electrophysiological and biochemical approaches, this study investigated the role of DOR83b in the olfactory system of Drosophila (Neuhaus, 2005).

Investigations of the function of DOR83b were done in a heterologous HEK293 expression system. Bioluminescence resonance energy transfer (BRET) experiments showed that DOR83b interacts with other odorant receptors. Energy transfer occurred when a DOR83b-luciferase (Luc) fusion construct was expressed together with DOR43a-green fluorescent protein (GFP) or with DOR22a-GFP in HEK293 cells, indicating that dimers or oligomers were forming between DOR83b and the conventional Drosophila odorant receptors DOR43a or DOR22a. The energy transfer was similar to the one measured with GFP and Luc-tagged β2-adrenergic receptors, G-protein-coupled receptors for which homodimerization is already known to occur. Similar results were obtained for coexpression of DOR83b-GFP and DOR83b-Luc and for coexpression of DOR43a-GFP and DOR43a-Luc, indicating that the receptors can also homodimerize. Receptor oligomerization was specific and not the result of receptor overexpression; no BRET was detected when DOR83b-Luc was coexpressed with the GFP-tagged cyclic nucleotide-gated channel from Drosophila or with the GFP-tagged rat β2-adrenergic receptor. Dimeric complexes persisted following denaturing gel electrophoresis and western blotting (Neuhaus, 2005).

To test the functional consequences of coexpression of DOR83b with DOR43a, calcium imaging measurements were performed in the heterologous HEK293 system, which has frequently been used to assay odorant receptor function. DOR43a can be activated by cyclohexanone, but in HEK293 cells responses were obtained only when ligand concentrations were in the millimolar range, and with poor efficiency (below 1% of cells responding). In contrast, when DOR43a was cotransfected with DOR83b, the threshold dropped to the micromolar range and the number of cells responding significantly increased to 10%-15%, even though expression of the receptors remained unchanged (30%-40% of cells after transfection). The DOR43a-DOR83b heterodimer had a spectrum of active ligands similar to that reported for DOR43a-expressing neurons and for recombinantly expressed DOR43a alone. Active substances were cyclohexanone, benzaldehyde, isoamyl acetate, cineole and cyclohexanol; inactive substances were ethylbutyrate, octanal and 2-octanal. To generalize these findings, the responses of DOR22a transfected alone and with DOR83b (DOR22a/DOR83b) was tested in HEK293 cells, and similar effects were found. DOR22a/DOR83b cotransfected cells responded to lower (micromolar compared to millimolar) concentrations of ethylbutyrate, one of the best ligands for DOR22a-expressing neurons, and the number of responding cells was significantly higher despite similar transfection rates. To check the specificity of the effect of the DOR83b cotransfection, DOR43a together with DOR22a was transfected into HEK293 cells and, again, obtained responses only at ligand concentrations in the millimolar range and with poor efficiency. DOR83b did not respond to any of the odorants tested, even in the millimolar range (Neuhaus, 2005).

If, as the BRET and calcium imaging data suggest, DOR83b functions as a co-receptor, a reduction in DOR83b expression in vivo should lead to a reduction in odor responsiveness of the ORNs. To investigate the influence of DOR83b expression in the fly, RNA interference was used to reduce the level of DOR83b, and the odor-evoked response of the antennae was monitored using electroantennogram (EAG) recordings. In flies hatched from embryos injected with DOR83b double-stranded RNA (RNAi flies), the EAG amplitude was reduced compared to control flies . Six different, commonly used odors evoked significantly smaller EAG responses in RNAi flies compared to control flies, with average reductions ranging from 56% for 1-butanol to 30% for ethanol. The residual response is likely to reflect incomplete knockdown of DOR83b mRNA, as suggested by the absence of detectable responses in DOR83b mutant flies. Moreover, in situ hybridization on the antennae of the flies (RNAi versus non-RNAi) on which EAG recordings had been done showed that DOR83b mRNA levels were much lower, but not completely abolished, in flies with lowered EAG responses (Neuhaus, 2005).

This study shows that insect odorant receptors form dimers and that heterodimerization improves the functionality of the receptors. The results suggest that DOR83b functions as a co-receptor of conventional Drosophila odorant receptors. Further investigation will be needed to determine whether dimerization is a general property of odorant receptors in vertebrates as well (Neuhaus, 2005).

Atypical membrane topology and heteromeric function of Drosophila odorant receptors in vivo

Drosophila olfactory sensory neurons (OSNs) each express two odorant receptors (ORs): a divergent member of the OR family and the highly conserved, broadly expressed receptor OR83b. OR83b is essential for olfaction in vivo and enhances OR function in vitro, but the molecular mechanism by which it acts is unknown. This study demonstrates that OR83b heterodimerizes with conventional ORs early in the endomembrane system in OSNs, couples these complexes to the conserved ciliary trafficking pathway, and is essential to maintain the OR/OR83b complex within the sensory cilia, where odor signal transduction occurs. The OR/OR83b complex is necessary and sufficient to promote functional reconstitution of odor-evoked signaling in sensory neurons that normally respond only to carbon dioxide. Unexpectedly, unlike all known vertebrate and nematode chemosensory receptors, Drosophila ORs and OR83b adopt a novel membrane topology with their N-termini and the most conserved loops in the cytoplasm. These loops mediate direct association of ORs with OR83b. These results reveal that OR83b is a universal and integral part of the functional OR in Drosophila. This atypical heteromeric and topological design appears to be an insect-specific solution for odor recognition, making the OR/OR83b complex an attractive target for the development of highly selective insect repellents to disrupt olfactory-mediated host-seeking behaviors of insect disease vectors (Benton, 2006).

These results define Drosophila OR83b and ORs as a novel family of TM proteins with sequence and membrane topology that is distinct from mammalian GPCR-family ORs. OR83b associates with ORs through conserved cytoplasmic loops previously believed to be extracellular; ORs and OR83b form heteromeric complexes within OSNs. These complexes form early in the membrane-trafficking pathways but persist and concentrate in the sensory cilia. An essential role is shown for OR83b in targeting and maintaining these complexes within the ciliary membranes at the site of odorant signal transduction. These results define OR83b as an integral part of the functional odorant receptor in insects. Furthermore, despite the striking similarities in the anatomy and physiology of mammalian and insect olfactory systems, they reveal important distinctions in the molecular nature of the odorant receptor in these organisms (Benton, 2006).

The role of ORs in translating the odorous environment into neuronal activity depends critically on their localization to the surface of the ciliated sensory endings of OSN dendrites. How ORs navigate from their site of synthesis in the ER to these specialized sensory compartments is poorly characterized but has long been suspected to depend upon olfactory-specific cofactors because of the difficulty in functionally expressing these proteins in heterologous cells. The data show that in insects an OR protein has been adapted to subserve a new cellular function: to traffic structurally similar ligand-binding ORs to olfactory cilia (Benton, 2006).

The observation that OR83b can localize to chemosensory cilia in the absence of associated ORs rules out the possibility that only the heteromeric OR/OR83b complex is transport-competent. Instead, the data indicate that OR83b itself can associate with the transport pathway in OSNs and functions to link ORs to this transport machinery. Because OR83b can promote OR localization to cilia in mechanosensory neurons, it likely couples to a general ciliary transport pathway, without the requirement for additional OSN-specific cofactors (Benton, 2006).

Analysis of the temporal requirement of OR83b also reveals an essential role for OR83b in maintaining ORs within this sensory compartment, since OR22a/b is never detected in cilia in the absence of OR83b. Together with observation of the persistence of OR/OR83b heteromeric complexes in the sensory compartment, these results strongly suggest that OR83b is an integral and stable component of the insect OR complex necessary for both proper localization and stability of the conventional ORs in dendrites, rather than a transient chaperone that shuttles and deposits monomeric ORs in the ciliary membrane (Benton, 2006).

While Drosophila ORs do not contain any known protein motifs, the strongest homology within members of this family spans predicted TM6 and TM7, suggesting that this region might mediate a function common to all ORs. Consistent with this conservation, it was found that the loop linking these predicted TM domains (IC3) forms at least part of the interaction interface between ORs and OR83b. In OR83b, this region is almost fully conserved between insect orthologues, and this may explain why OR83b orthologues from diverse insects can functionally substitute for OR83b in Drosophila. Comparison of OR83b with other ORs reveals the presence of a 70-amino-acid insertion in IC2 that is unique to OR83b. The data suggest this loop is located in the cytoplasm, and it is speculated that the insertion links OR83b to the intracellular transport machinery (Benton, 2006).

Most mammalian ORs fail to reach the cell surface when expressed in heterologous cells but are largely trapped in aggregates in the ER and are eventually degraded. This is remarkably similar to the fate of Drosophila ORs in Or83b mutants. OR trafficking in mammals appears to have been solved differently from insects. Screens for olfactory-specific genes that facilitate OR localization have identified a number of single TM domain accessory factors that associate with but are structurally unrelated to the ORs: REEP, RTP1, and RTP2 for mouse ORs and MHC class 1b proteins for mouse V2R pheromone receptors (Loconto, 2003; Saito, 2004). A single TM domain protein, ODR-4, has also been shown to be required for OR trafficking in C. elegans but, unlike OR83b, ODR-4 is not present in sensory cilia (Benton, 2006 and references therein).

Heterodimerization of structurally related chemosensory receptors is important for mammalian taste perception, but this modulates the ligand-binding properties of these receptors rather than their subcellular localization. Thus, the requirement for a universal co-receptor for chemoreception appears to be unique to the Drosophila OR family, and this relies on the remarkable property of OR83b to couple both to the conserved ciliary trafficking machinery and probably to all 61 members of the highly divergent OR family (Benton, 2006).

The presence of OR/OR83b complexes in the sensory compartment raises the possibility that OR83b has additional functions in olfactory perception. This is difficult to address in vivo because of the essential requirement for OR83b in OR localization. However, the diversity in odor response profiles of different classes of OR83b-expressing neurons makes it unlikely that OR83b itself recognizes ligands. This has been confirmed experimentally in Or22a/b mutant neurons, which do not respond to odors even though OR83b is present in these sensory cilia. In vitro studies have revealed that some ectopically expressed ORs in heterologous cells are capable of recognizing odors in the absence of OR83b, albeit with low efficiency. The odor-response profiles of these ORs is similar to that observed in vivo, which suggests that OR83b is unlikely to influence OR ligand specificity but could facilitate efficient ligand-receptor interactions by, for example, maintaining the conformation of ORs within the ciliary membrane (Benton, 2006).

Although the site of ligand interaction in insect ORs and GRs is unknown, a naturally occurring single amino acid polymorphism (A218T) in GR5a influences the sensitivity of this receptor to trehalose. Previously, this residue was thought to lie in the second intracellular loop, where it was proposed to affect coupling of GR5a to G proteins. In the topology model for insect chemosensory receptors proposed here, this residue is predicted to be extracellular and may instead influence ligand binding (Benton, 2006).

The molecular components of primary olfactory signal transduction pathways in insects are unknown. However, in vivo misexpression experiments indicate that ORs can function in other OR-expressing OSNs. This suggests that ORs converge on a common signaling cascade in Or83b neurons, and it is attractive to suggest that OR83b might form part of this common pathway. The capacity of OR83b to couple to downstream signal transduction components is supported by the observation that OSNs expressing OR83b but lacking conventional ORs show spontaneous activity while Or83b mutant OSNs do not. The demonstration that OR/OR83b complexes can function in Gr21a neurons suggests that OR83b may not define a signal transduction pathway that is unique to OR-expressing sensory neurons, but rather that the divergent OR and GR chemosensory receptor families can couple to a common cascade (Benton, 2006).

The structural distinction between insect and mammalian ORs begs the question of whether G proteins are involved in insect chemosensory signaling. In both mammals and C. elegans, loss-of-function genetics provides strong support for a role of G proteins in olfactory signal transduction. In contrast, no direct experimental evidence exists for G protein signaling downstream of insect ORs. Several G alpha subunits, in particular Gαq, are expressed in insect antennae, but they are not specifically enriched in the ciliated dendrite of OSNs. Reduction of Gαq levels in Drosophila OSNs produces defective behavioral responses to some odor stimuli, although it is not known whether this is due to a primary defect in olfactory signal transduction. Surprisingly, OR/OR83b odor-evoked signaling is observed in heterologous cells with or without co-expression of exogenous insect Gαq proteins, suggesting that these proteins have the capacity to couple to endogenous, but unknown, signaling molecules. Thus, despite the widespread assumption that insect chemoreception employs a canonical G protein-signaling cascade, the evidence in support of this is inconclusive (Benton, 2006).

While defining the molecular nature of insect olfactory signaling remains an important goal, the observation that insect ORs are structurally unrelated to the GPCR superfamily raises two equally intriguing possibilities. Insect chemosensory receptors may represent a second family of polytopic TM proteins that has evolved independently to couple to G proteins. Alternatively, these receptors may not couple to G proteins but activate a distinct signaling cascade in response to odor stimulation (Benton, 2006).

While ORs in mammals and insects share a common function in translating odor stimuli into neuronal activity, these findings reveal fundamental differences in the molecular basis of olfactory perception in these organisms. That their OR families should have unrelated evolutionary origins highlights the remarkable convergence in the anatomical and physiological mechanisms that mammals and insects display in the representation of odors in their peripheral circuits. This work raises important questions about the mechanism of odor recognition and olfactory signal transduction in insects. Furthermore, the demonstration that the OR/OR83b complex is the essential molecular unit of olfactory perception in insects makes this complex an attractive target for the development of highly selective insect repellents to interrupt chemosensory-driven, host-seeking behaviors of insect vectors of human disease (Benton, 2006).

Phosphorylation via PKC regulates the function of the Drosophila odorant co-receptor

Insect odorant receptors (ORs) have a unique design of heterodimers formed by an olfactory receptor protein and the ion channel Orco. Heterologously expressed insect ORs are activated via an ionotropic and a metabotropic pathway that leads to cAMP production and activates the Orco channel. The contribution of metabotropic signaling to the insect odor response remains to be elucidated. Disruption of the Gq protein signaling cascade has been shown to reduce the odor response. This study investigated this phenomenon in HEK293 cells expressing Drosophila Orco and found that phospholipase C (PLC) inhibition reduced the sensitivity of Orco to cAMP. A similar effect was seen upon inhibition of protein kinase C (PKC), whereas PKC stimulation activated Orco even in the absence of cAMP. Mutation of the five PKC phosphorylation sites in Orco almost completely eliminated sensitivity to cAMP. To test the impact of PKC activity in vivo, single sensillum electrophysiological recordings were combined with microinjection of agents affecting PLC and PKC function, and an altered response of olfactory sensory neurons (OSNs) to odorant stimulation was observed. Injection of the PLC inhibitor U73122 or the PKC inhibitor Go6976 into sensilla reduced the OSN response to odor pulses. Conversely, injection of the PKC activators OAG, a diacylglycerol analog, or phorbol myristate acetate (PMA) enhanced the odor response. It is concluded that metabotropic pathways affecting the phosphorylation state of Orco regulate OR function and thereby shape the OSN odor response (Sargsyan, 2011).

Dimerisation of the Drosophila odorant coreceptor Orco

Odorant receptors (ORs) detect volatile molecules and transform this external information into an intracellular signal. Insect ORs are heteromers composed of two seven transmembrane proteins, an odor-specific OrX and a coreceptor (Orco) protein. These ORs form ligand gated cation channels that conduct also calcium. The sensitivity of the ORs is regulated by intracellular signaling cascades. Heterologously expressed Orco proteins form also non-selective cation channels that cannot be activated by odors but by synthetic agonists such as VUAA1. The stoichiometry of OR or Orco channels is unknown. This study engineered the simplest oligomeric construct, the Orco dimer (Orco di) and investigated its functional properties. Two Orco proteins were coupled via a 1-transmembrane protein to grant for proper orientation of both parts. The Orco di construct and Orco wild type (Orco wt) proteins were stably expressed in CHO (Chinese Hamster Ovary) cells. Their functional properties were investigated and compared by performing calcium imaging and patch clamp experiments. With calcium imaging experiments using allosteric agonist VUAA1 it was demonstrated that the Orco di construct-similar to Orco wt-forms a functional calcium conducting ion channel. This was supported by patch clamp experiments. The function of Orco di was seen to be modulated by CaM in a similar manner as the function of Orco wt. In addition, Orco di interacts with the OrX protein, Or22a. The properties of this complex are comparable to Or22a/Orco wt couples. Taken together, the properties of the Orco di construct are similar to those of channels formed by Orco wt proteins. These results are thus compatible with the view that Orco wt channels are dimeric assemblies (Mukunda, 2014).


DEVELOPMENTAL BIOLOGY

The expression of Or83bhas been described only anecdotally (Vosshall, 1999; Vosshall, 2000): a detailed characterization of the distribution and subcellular localization of this protein is presented in this study. The fly has olfactory sensory neurons (OSNs) in three specialized structures: the dorsal organ of the larva and the maxillary palps and third antennal segments on the adult head. Or83b expression is restricted to OSNs and is not detected in other tissues, including gustatory neurons. The expression of the Or83b mRNA was examined throughout the four main developmental stages of Drosophila. Or83b is first detected late in embryonic development at stage 15, where its expression is limited to the antennal-maxillary complex, an anterior structure that contains the OSNs. Later in development, it is expressed in all 21 OSNs of the larval dorsal organ located at the anterior tip of the larva. During metamorphosis, all of these larval OSNs are destroyed and replaced by new OSNs that populate the antenna and maxillary palp. In the pupa, Or83b is first detected in antennal OSNs at 80 hr after puparium formation, which is late in pupal development and approximately coincident with the onset of expression of conventional OR genes. Or83b expression in all adult maxillary palp neurons and ~70%-80% of antennal OSNs. The exact proportion of neurons that express Or83b in the antenna is not known and is difficult to estimate because Or83b is expressed at different levels in different subpopulations of OSNs. Levels are highest at the dorsal-medial region (Larsson, 2004).

Both in the antenna and maxillary palp, each Or83b positive OSN expresses Or83b along with at least one conventional OR gene. Or22a/band Or83b mRNA overlap in a group of dorsal-medial neurons. An anti-Or83b antibody was raised against loop residues between transmembrane domains three and four and this antibody was used to determine the subcellular localization of Or83b. Or83b protein is found in ciliated OSN dendrites, the chemosensory specialization in which OR proteins are localized and where olfactory signal transduction occurs. Double immunostaining with a rabbit anti-Or22a/b antibody and a mouse anti-Or83b antibody demonstrates that these two OR proteins colocalize in the distal portion of the dendrite inserted into the sensory hair. Or83b is also detected in OSN cell bodies but is not seen in proximal axons in the antenna or at axonal termini in the antennal lobe of the brain, suggesting that its function lies in the ligand-detecting region of these polarized sensory neurons (Larsson, 2004).

To examine the connectivity of Or83b-expressing neurons, an Or83b promoter-enhancer trans-gene (Wang, 2003) in combination with the synaptic marker, n-synaptobrevin-Green Fluorescent Protein was used to visualize projections of these neurons in the antennal lobe. To do this, advantage was taken of the two-component gene regulation system that uses the yeast Gal 4 transcription factor fused to the Or83b regulatory regions and flies carrying this driver transgene were crossed to a UAS-nsyb-GFP responder transgene. Consistent with the apparent medial to lateral gradient of Or83b expression in the antenna, there is a similar gradient of fibers innervating the antennal lobe. Dorsal and medial glomeruli, including those that receive input from Or22a/b-expressing OSNs were brightly labeled. Lateral glomeruli receive considerably less innervation. Confocal sections through central and posterior aspects of the antennal lobe clearly show the sparsely innervated lateral and ventral glomeruli. Notably, the V glomerulus receives no apparent input from Or83b-expressing neurons (Larsson, 2004).


EFFECTS OF MUTATION

To study the loss of function of Or83b, null mutants were generated in which the putative transcription start site and the first five transmembrane domains of this seven transmembrane domain receptor protein were deleted by gene-targeting techniques designed to replace these portions of the Or83b coding region with the white gene, a selectable eye color marker. 14 homozygous mutants were obtained and confirmed to be null by Southern blotting with probes that lie both 5' and 3' of the targeted locus and by PCR directed against the Or83b coding region. The homozygous mutants produce viable and fertile adult progeny with no gross developmental or physical defects. Or83b mutant larvae show normal locomotor and gustatory behavior. Three mutant lines (Or83b1, Or83b2, Or83b3) were selected for further characterization, in addition to a genetically matched control line in which the targeting construct integrated but left the Or83b region intact (Larsson, 2004).

No Or83b mRNA or Or83b protein is detectable in the homozygous Or83b2 mutant strain in Or83b-expressing neurons. Antibodies directed against the terminal cytoplasmic loop of Or83b, encoded by DNA sequences left intact by the targeted deletion, also show no detectable residual Or83b protein. This rules out the possibility that partial expression of a truncated protein could interfere with mutant analysis by causing dominant-negative effects. Similar expression results were obtained with the Or83b2 and Or83b3 strains (Larsson, 2004).

In animals carrying an Or83b rescuing transgene under the control of the Or83b-Gal4 driver, Or83b expression is partially restored. The medial to lateral gradient of Or83b expression is exaggerated in these animals, producing lower than normal levels of Or83b expression in the lateral-distal domains of the antenna. Rescue of Or83b expression in mutants under control of this transgenic driver in larvae also show lower than wild-type levels in certain dorsal organ neurons (Larsson, 2004).

Because Or83b is broadly coexpressed with other ORs, it was asked whether the loss of Or83b function has an effect on the distribution or levels of expression of other coexpressed ORs. In adult olfactory neurons, both Or22a/b and Or43b proteins localize to OSN dendrites in wild-type antenna. Or22a/b is particularly efficiently transported into the dendrite with little protein detected in the cell body, while Or43b is distributed in a manner similar to Or83b in both sensory dendrite and cell body. In all three Or83b mutants both Or22a/b and Or43b proteins are weakly detected in the cell body but no dendritic staining is seen. The available reagents do not permit determination of whether residual Or22a/b and Or43b proteins are intracellular or on the surface of the cell body in Or83b mutants. In control experiments, no signal is detected with these antibodies in Or22a/b and Or43b null mutants. Restoring Or83b gene function by transgenic rescue restores the subcellular localization of both Or22a/b and Or43b to the chemosensory dendrite (Larsson, 2004).

Because odorant binding and signal transduction occurs in the dendrite, the OR mislocalization defect found in Or83b mutants would be expected to disrupt odor-evoked physiology. It is therefore of great importance to determine whether this failure of conventional OR proteins to be localized in the dendrite reflects a specific requirement of Or83b or is a nonspecific effect of a sick neuron. Therefore a number of control experiments were carried out to determine whether other aspects of the biology of OSNs is normal in Or83b mutants. The stereotyped targeting of Or22a-expressing axons to the DM2 glomerulus in the antennal lobe is not affected in the mutant. Or22a-nsyb-GFP-positive fibers are found restricted to only the DM2 glomerulus, and no ectopic innervation is seen. Levels of this synaptic marker are considerably lower in the mutant than in wild-type, which likely is a direct consequence of the disruption of neuronal activity in these neurons. Similar results were obtained on reduced trafficking of both membrane-targeted and synaptically targeted marker proteins in neurons that have been silenced genetically, perhaps because of a general effect on axonal and synaptic membrane protein transport in inactive neurons. This suggests that Or83b is not required for the establishment or maintenance of synaptic connections in glomeruli and that these connections persist in the absence of neuronal activity. The gross morphology of OSNs is normal in Or83b mutants, as judged by the levels and distribution of the neuron-specific microtubule associated protein, Futsch, recognized by the 22c10 monoclonal antibody. In an additional control experiment, a mix of antisense DOR gene probes was hybridized to wild-type and mutant antennae and it was found that the numbers, distribution, and levels of gene expression of these OR genes are comparable in both genotypes (Larsson, 2004).

Finally, the subpopulation of Or22a/b neurons was electrically silenced in wild-type animals to determine if loss of neuronal activity per se affects the accumulation or maintenance of Or22a/b and Or83b protein in dendrites. Expression of the inward rectifying potassium channel Kir2.1 in Or83b-expressing neurons eliminates EAG responses to all odors tested. However, normal dendritic localization of Or22a/b and Or83b is maintained under conditions of prolonged electrical silencing that mimic other aspects of the Or83b phenotype, including the reduced levels of synaptic markers in OSN axonal termini This suggests that the dendritic localization defect seen in Or83b animals reflects a specific role of this gene in protein localization and is not a secondary consequence of a loss of neuronal activity (Larsson, 2004).

The effect of the Or83b mutation on OR localization in the 21 neurons of the larval dorsal organ was investigated. These neurons are clustered into a ganglion containing the cell bodies, each of which extends a dendrite that inserts into the dorsal organ dome. Cytoplasmic lacZ marker protein labels the cell bodies and basal portions of the dendrites but is excluded from the distal tip of the dendrite inserted in the dome. The Or83b-Gal4 driver was used to misexpress an adult OR, Or22a, in all larval OSNs. Or22a protein is localized to the distal dendritic tips as seen in an exterior view of the dorsal organ dome. Lower levels of Or22a are detected in the cell bodies (Larsson, 2004).

In Or83b mutant larvae, no Or22a protein is detected in dendrites and low levels are seen in the cell bodies. In wild-type animals, Or83b protein is detected in distal dendritic tips inserted into the dorsal organ dome as well as the OSN cell bodies but is not detected in Or83b mutants. The accumulation of the membrane bound marker CD8-GFP in the distal dorsal organ dendrites is maintained in Or83b mutant OSNs, ruling out a general defect in dendritic morphology or protein sorting in the mutants. Therefore, larval OSNs have the same requirement for Or83b gene function in localizing ORs to the site of odor interaction in dendrites as adult OSNs (Larsson, 2004).

The dramatic and selective effect of the Or83b mutation on OR localization in olfactory dendrites would be expected to eliminate odor-evoked potentials in these neurons. To test this, electroantennograms (EAGs), which are thought to reflect broad domains of receptor potentials summed around the site of the recording electrode in the fly antenna, were measured. Control preparations show strong negative potentials in response to all odorants tested. EAGs recorded in Or83b mutant antennae produce no responses, and normal EAG responses are restored by transgenic rescue of Or83b gene function. These results are consistent with a general requirement for Or83b in adult olfactory function to a broad range of odor stimuli (Larsson, 2004).

EAGs provide a convenient measure of olfactory function across the entire antenna but suffer from a lack of single cell resolution and have low sensitivity. Therefore, to be certain that this mutation blocks odor-evoked activity at the single neuron level, extracellular single sensillum recordings were carried out on the identified ab1 sensillum, which contains three general odor-responsive neurons and a single neuron (ab1C) highly tuned to carbon dioxide. In wild-type antennae, all four neurons in the ab1 sensillum show characteristic odor responsivity. Or83b mutant antennae show no odor-evoked action potentials to the general odorants ethyl acetate (ab1A), acetoin (ab1B), or methyl salicylate (ab1D), but responses to carbon dioxide (ab1C) are completely normal. Unlike the case of neurons with deficiencies in conventional odorant receptors, Or83b mutant OSNs generally show very little spontaneous activity. Evidence of spontaneous or damage-induced activity of the general odor neurons were occasionally found in these animals, which may reflect low levels of spontaneous activity independent of receptor activation. Restoring Or83b gene function to antennal neurons by transgenic rescue also restores wild-type odor responses to the three affected neurons of the ab1 sensillum (Larsson, 2004).

The dramatic loss of odor-evoked potentials to a broad range of general odors at the level of the whole antenna and single neurons suggests a general and essential role for Or83b in odor detection. To determine the behavioral effects of this mutation on larval behavior, chemotaxis was monitored with a single-animal video-tracking assay, modified from a previously described population assay. Tracks of animals responding to ethyl acetate were examined. While the control yw animal chemotaxes toward the stimulus, the Or83b1 mutant larva does not respond to the odor and wanders at random around the plate. Expression of a rescuing UAS-Or83b transgene under control of the Or83b-Gal4 driver in the Or83b1 mutant restores chemotaxis behavior. Olfactory responses of animals with these three genotypes were tested to 36 odors. Control yw larvae chemotax to all 36 odors, while mutant larvae showed no response to 34 of these odors and weak responses to 2. The Or83b rescuing transgene partially restores chemotaxis in Or83b1 mutants, consistent with the nonuniform expression of the Or83b-Gal4 driver in the larval dorsal organ. Similar results were obtained with two additional mutant and control strains to a subset of these odors (Larsson, 2004).

To determine whether the electrophysiological defects seen in the antenna translate to behavioral phenotypes, a modified trap assay was performed. Adult flies show strong behavioral responses to natural odors and synthetic odor blends but respond to only a subset of the single odors composing these blends. The chemosensory behavior of adult Or83b mutant flies was tested with two of these behaviorally active single odors, acetoin and 2-phenylethanol. As expected from the absence of odor-evoked physiological responses to these odors, Or83b mutants show severely impaired responses to acetoin and are anosmic to 2-phenylethanol. Normal responses to acetoin and 2-phenylethanol are restored in Or83b2 mutants carrying a UAS-Or83b rescuing transgene under control of the Or83b-Gal4 driver (Larsson, 2004).

Learned odor discrimination in Drosophila without combinatorial odor maps in the antennal lobe

A unifying feature of mammalian and insect olfactory systems is that olfactory sensory neurons (OSNs) expressing the same unique odorant-receptor gene converge onto the same glomeruli in the brain. Most odorants activate a combination of receptors and thus distinct patterns of glomeruli, forming a proposed combinatorial spatial code that could support discrimination between a large number of odorants. OSNs also exhibit odor-evoked responses with complex temporal dynamics, but the contribution of this activity to behavioral odor discrimination has received little attention. This study investigated the importance of spatial encoding in the relatively simple Drosophila antennal lobe. Flies can learn to discriminate between two odorants with one functional class of Or83b-expressing OSNs. Furthermore, these flies distinguish one odorant from a mixture and cross-adapt to odorants that activate the relevant OSN class, demonstrating that they discriminate odorants by using the same OSNs. Lastly, flies with a single class of Or83b-expressing OSNs recognize a specific odorant across a range of concentration, indicating that they encode odorant identity. Therefore, flies can distinguish odorants without discrete spatial codes in the antennal lobe, implying an important role for odorant-evoked temporal dynamics in behavioral odorant discrimination (DasGupta, 2008).

In conclusion, multiple classes of OSNs are not required for flies to discriminate odorants. Although flies without functional Or83b-expressing neurons cannot learn to discriminate between a number of chemically distinct odorants, providing a single class of Or83b-expressing OSNs restores learned discrimination between two odorants that activate that particular OSN class. These flies cross-adapt to odorants that activate the restored OSNs, demonstrating that the relevant OSNs are the same, thus challenging a requirement for discrete spatial codes for odorants in the antennal lobe. As expected, flies with one class of Or83b-expressing OSNs have limitations and can apparently only encode one odorant receptor that activates the appropriate receptor at a time. These data suggest that a benefit of having multiple classes of OSNs is the ability to identify certain odorants present within a more complex milieu. Importantly, Or83b2 mutant flies with one functional class of Or83b-expressing OSNs choose appropriately between two odorants even though the absolute and relative concentration was changed between training and testing, implying that they encode odorant identity and do not only rely on encoding odorant intensity (DasGupta, 2008).

Finding that distinct combinatorial spatial patterns of OSN activation in the antennal lobe are not essential to represent odorant information implies an important role for odorant-evoked temporal dynamics. Previous studies in insects and vertebrates have documented considerable temporal complexity in odor-evoked activity at successive layers of the olfactory system, but few have investigated the behavioral relevance. Recent work has shown that excitatory and inhibitory lateral connectivity in the Drosophila antennal lobe can shape projection neuron responses; therefore, it can be expected that different temporal signals in the same OSNs generate distinct temporal, and perhaps spatial, patterns of projection neuron activity. However, because flies with a single functional class of Or83b-expressing OSNs lack the lateral input driven by additional classes of OSN, it will be important to determine how lateral connectivity within the antennal lobe contributes to odorant discrimination in Drosophila (DasGupta, 2008).

Drosophila TRPA1 channel is required to avoid the naturally occurring insect repellent citronellal

Plants produce insect repellents, such as citronellal, which is the main component of citronellal oil. However, the molecular pathways through which insects sense botanical repellents are unknown. This study shows that Drosophila uses two pathways for direct avoidance of citronellal. The olfactory coreceptor OR83b contributes to citronellal repulsion and is essential for citronellal-evoked action potentials. Mutations affecting the Ca2+-permeable cation channel TRPA1 result in a comparable defect in avoiding citronellal vapor. The TRPA1-dependent aversion to citronellal relies on a G protein (Gq)/phospholipase C (PLC) signaling cascade rather than direct detection of citronellal by TRPA1. Loss of TRPA1, Gq, or PLC causes an increase in the frequency of citronellal-evoked action potentials in olfactory receptor neurons. Absence of the Ca2+-activated K+ channel (BK channel) Slowpoke results in a similar impairment in citronellal avoidance and an increase in the frequency of action potentials. These results suggest that TRPA1 is required for activation of a BK channel to modulate citronellal-evoked action potentials and for aversion to citronellal. In contrast to Drosophila TRPA1, Anopheles gambiae TRPA1 is directly and potently activated by citronellal, thereby raising the possibility that mosquito TRPA1 may be a target for developing improved repellents to reduce insect-borne diseases such as malaria (Kwon, 2010).

Two features of the citronellal responses were found to be abnormal in the trpA11 basiconic sensilla ab11a neurons. First, there was a higher citronellal-evoked action potential frequency than in wild-type. Second, there was a defect in deactivation in trpA11 ab11a neurons. The same two defective phenotypes were observed in ab11 neurons in the dGqα1 and norpAP24 mutants, although only the increase in the evoked responses was clearly different when testing significance by analysis of variance. These results support the conclusion that the dGqα1, norpAP24, and trpA11 mutations affect the citronellal response in an ORN in ab11 (Kwon, 2010).

The finding that there were increases in the frequency of citronellal-evoked action potentials was unexpected and raised a question as to the basis for these defects. TRPA1 is a Ca2+-permeable channel, and because reduced activity of Ca2+-activated K+ channels (BK channels) increases the frequency of action potential firing, it was of interest to see whether loss of TRPA1 caused reduced BK channel activity. If so, then a mutation in the gene (slowpoke, slo) encoding the fly BK channel might phenocopy the trpA1 phenotype. In support of this model, the slof05915 mutation caused an increase in the frequency of citronellal-evoked action potentials and impaired citronellal avoidance. Introduction of UAS-slo-RNAi in combination with either the trpA1-GAL4 or the Or83b-GAL4 resulted in a similar defect in citronellal avoidance (Kwon, 2010).

It is proposed that TRPA1 is required for activity of Slo, which in turn modulates citronellal-induced firing of action potentials. Slo might be required in many ORNs and be regulated by additional TRP channels. In support of this proposal, ab12 also responded to citronellal and displayed a higher frequency of action potentials in slof05915 but did not function through a Gqα/PLC/TRPA1 pathway. Knockout of a mammalian TRP channel, TRPC1, also disrupts the activity of a Ca2+-activated K+ channel (KCa) in salivary gland cells, and mutations affecting either TRPC1 or KCa result in similar defects in salivary gland secretion. Thus, a role for TRP channels in activating Ca2+-activated K+ channels might be a common but poorly appreciated general phenomenon that is evolutionarily conserved (Kwon, 2010).

The finding that loss of TRPA1 causes an increase rather than a decrease in citronellal-induced action potentials suggests that there might be a TRPA1 independent-pathway required for generating action potentials in response to citronellal. OR83b is a candidate for functioning in such a pathway, because mutation of Or83b interferes with the ability of the synthetic repellent DEET to inhibit the attraction to food odors. This study found that Or83b1 mutant flies, or Or83b1 in trans with a deficiency that uncovers the locus, exhibited an impairment in citronellal avoidance similar to that in trpA1 mutant flies. An Or83b1 defect in the DART assay was not specific to citronellal, because these flies were also impaired in the response to benzaldehyde. Tested were performed to see whether the frequency of citronellal-induced action potentials was altered in Or83b1 ab11 sensilla. In contrast to the trpA1 mutant phenotype, none of the mutant Or83b1 ab11 neurons responded to citronellal (Kwon, 2010).

These data indicate that there are dual pathways required for the response to citronellal. OR83b is necessary for producing citronellal-induced action potentials, and a Gq/PLC/TRPA1 pathway appears to function in the modulation of action potential frequency by activating BK channels. It is suggested that an abnormally high frequency of action potentials may lead to rapid depletion of the readily releasable pools of neurotransmitter, thereby muting the citronellal response. Interestingly, a loss-of-function mutation affecting a worm BK channel also results in a behavioral phenotype—increased resistance to ethanol. Although Drosophila TRPA1 functions downstream of a Gq/PLC signaling pathway, citronellal can also directly activate TRPA1, but with low potency. Nevertheless, because Anopheles gambiae TRPA1 is also expressed in the antenna and is activated directly by citronellal with high potency, it is suggested that mosquito TRPA1 represents a new potential target for in vitro screens for volatile activators that might serve as new types of insect repellents (Kwon, 2010).

Drosophila tracks carbon dioxide in flight

Carbon dioxide (CO2) elicits an attractive host-seeking response from mosquitos yet is innately aversive to Drosophila melanogaster despite being a plentiful byproduct of attractive fermenting food sources. Prior studies used walking flies exclusively, yet adults track distant food sources on the wing. This study shows that a fly tethered within a magnetic field allowing free rotation about the yaw axis actively seeks a narrow CO2 plume during flight. Genetic disruption of the canonical CO2-sensing olfactory neurons does not alter in-flight attraction to CO2; however, antennal ablation and genetic disruption of the Ir64a acid sensor do. Surprisingly, mutation of the obligate olfactory coreceptor (Orco; Or83b) does not abolish CO2 aversion during walking yet eliminates CO2 tracking in flight. The biogenic amine octopamine regulates critical physiological processes during flight, and blocking synaptic output from octopamine neurons inverts the valence assigned to CO2 and elicits an aversive response in flight. Combined, these results suggest that a novel Orco-mediated olfactory pathway that gains sensitivity to CO2 in flight via changes in octopamine levels, along with Ir64a, quickly switches the valence of a key environmental stimulus in a behavioral-state-dependent manner (Wasserman, 2013).

These results show that a single molecule can carry both negative and positive hedonic valence depending on the behavioral state of the animal. It is posited that flight behavior is accompanied by neuromodulatory activation of the olfactory system by octopamine that rapidly shifts the function of olfactory sensory pathways in a manner similar to the operational gain and frequency response shifts triggered by locomotor activity in fly visual interneurons. Recent work in other organisms has identified similar roles for neuromodulators that serve to alter the state of neuronal circuits in a behaviorally contextual manner, thereby enabling computational flexibility and behavioral robustness to ever-changing internal and external environmental conditions. These findings unravel the paradox of why D. melanogaster would find an environmental signal indicating a potential food source repellent instead of attractive; for Drosophila gathered on the ground, under crowded social conditions, CO2 secreted as part of a stress pheromone releases an innate avoidance response. Taking flight appears to fully and rapidly switch the valence of this stimulus, triggering CO2 attraction consistent with the search for sugar-rich food resources undergoing fermentation that robustly attract D. melanogaster vinegar flies. These findings lay the groundwork for further exploring the neural substrate for a rapid and robust switch in hedonic valence (Wasserman, 2013).

The neuro-ecology of Drosophila pupation behavior

Many species of Drosophila form conspecific pupa aggregations across the breeding sites. These aggregations could result from species-specific larval odor recognition. To test this hypothesis larval odors of D. melanogaster and D. pavani, two species that coexist in the nature, were tested. When stimulated by those odors, wild type and vestigial (vg) third-instar larvae of D. melanogaster pupated on conspecific larval odors, but individuals deficient in the expression of the odor co-receptor Orco randomly pupated across the substrate, indicating that in this species, olfaction plays a role in pupation site selection. Larvae are unable to learn but can smell, the Synapsin (Syn97CS) and rut strains of D. melanogaster, did not respond to conspecific odors or D. pavani larval cues, and they randomly pupated across the substrate, suggesting that larval odor-based learning could influence the pupation site selection. Thus, Orco, Syn97CS and rut loci participated in the pupation site selection. When stimulated by conspecific and D. melanogaster larval cues, D. pavani larvae also pupated on conspecific odors. The larvae of D. gaucha, a sibling species of D. pavani, did not respond to D. melanogaster larval cues, pupating randomly across the substrate. In nature, D. gaucha is isolated from D. melanogaster. Interspecific hybrids, which result from crossing pavani female with gaucha males clumped their pupae similarly to D. pavani, but the behavior of gaucha female x pavani male hybrids was similar to D. gaucha parent. The two sibling species show substantial evolutionary divergence in organization and functioning of larval nervous system. D. melanogaster and D. pavani larvae extracted information about odor identities and the spatial location of congener and alien larvae to select pupation sites. It is hypothesized that larval recognition contributes to the cohabitation of species with similar ecologies, thus aiding the organization and persistence of Drosophila species guilds in the wild (Del Pino, 2014).

Mutational analysis of cysteine residues of the insect odorant co-receptor (Orco) from Drosophila melanogaster reveals differential effects on agonist- and odorant/tuning receptor-dependent activation

Insect odorant receptors are heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor (ORx) and a highly conserved co-receptor known as Orco. Orco is found only in insects and very little is known about its structure and the mechanism leading to channel activation. In the absence of an ORx, Orco forms homomeric channels that can be activated by a synthetic agonist, VUAA1. Drosophila melanogaster Orco (DmelOrco) contains 8 cysteine amino acid residues, 6 of which are highly conserved. Individual cysteine residues were replaced with serine or alanine, and Orco mutants were expressed in FlpIn 293 T-Rex cells. Changes in intracellular Ca2+ levels were used to determine responses to VUAA1. Replacement of two cysteines (C429 and C449) in a predicted intracellular loop (ICL3), individually or together, gave variants that all showed similar increases in the rate of response and sensitivity to VUAA1 compared with wild-type Orco. Kinetic modelling indicated that the response of the Orco mutants to VUAA1 was faster than wild-type Orco. The enhanced sensitivity and faster response of the cys-mutants was confirmed by whole-cell voltage-clamp electrophysiology. In contrast to the results from direct agonist activation of Orco the two cysteine replacement mutants when co-expressed with a tuning receptor (OR22a) showed an approximately ~10-fold decrease in potency for activation by 2-methyl hexanoate. This work has shown that intracellular loop 3 is important for Orco channel activation. Importantly, this study also suggests differences in the structural requirements for the activation of homomeric and heteromeric Orco channel complexes (Turner, 2014).


EVOLUTIONARY HOMOLOGS

A candidate olfactory receptor subtype highly conserved across different insect orders

Candidate olfactory receptors of the moth Heliothis virescens were found to be extremely diverse from receptors of the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae, but there is one exception. The moth receptor type HR2 shares a rather high degree of sequence identity with one olfactory receptor type both from Drosophila (Dor83b) and from Anopheles (AgamGPRor7); moreover, in contrast to all other receptors, this unique receptor type is expressed in numerous antennal neurons. This study describes the identification of HR2 homologues in two further lepidopteran species, the moths Antheraea pernyi and Bombyx mori, which share 86%-88% of their amino acids. In addition, based on RT-PCR experiments HR2 homologues were discovered in antennal cDNA of the honey bee (Apis mellifera; Hymenoptera), the blowfly (Calliphora erythrocephala; Diptera) and the mealworm (Tenebrio molitor; Coleoptera). Comparison of all HR2-related receptors reveals a high degree of sequence conservation across insect orders. In situ hybridization of antennal sections from the bee and the blowfly support the notion that HR2-related receptors are generally expressed in a very large number of antennal cells. This, together with the high degree of conservation suggests that this unique receptor subtype may fulfill a special function in chemosensory neurons of insects (Krieger, 2003).

A highly conserved candidate chemoreceptor expressed in both olfactory and gustatory tissues in the malaria vector

Anopheles gambiae is a highly anthropophilic mosquito responsible for the majority of malaria transmission in Africa. The biting and host preference behavior of this disease vector is largely influenced by its sense of smell, which is presumably facilitated by G protein-coupled receptor signaling. Because of the importance of host preference to the mosquitoes' ability to transmit disease, studies have been intended to elucidate the molecular mechanisms underlying olfaction in An. gambiae. In the course of these studies, a number of genes were identified potentially involved in signal transduction, including a family of candidate odorant receptors. One of these receptors, encoded by GPRor7 (hereafter referred to as AgOr7), is remarkably similar to an odorant receptor that is expressed broadly in olfactory tissues and has been identified in Drosophila melanogaster and other insects (See alignment in Pitts, 2004). AgOr7 expression is observed in olfactory and gustatory tissues in adult An. gambiae and during several stages of the mosquitoes' development. Within the female adult peripheral chemosensory system, antiserum against the AgOR7 polypeptide labels most sensilla of the antenna and maxillary palp as well as a subset of proboscis sensilla. Furthermore, AgOR7 antiserum labeling is observed within the larval antenna and maxillary palpus. These results are consistent with a role for AgOr7 in both olfaction and gustation in An. gambiae and raise the possibility that AgOr7 orthologs may also be of general importance to both modalities of chemosensation in other insects (Pitts, 2004).

Functional agonism of insect odorant receptor ion channels.

In insects, odor cues are discriminated through a divergent family of odorant receptors (ORs). A functional OR complex consists of both a conventional odorant-binding OR and a nonconventional coreceptor (Orco) that is highly conserved across insect taxa. Recent reports have characterized insect ORs as ion channels, but the precise mechanism of signaling remains unclear. This study reports the identification and characterization of an Orco family agonist, the chemical compound VUAA1, using the Anopheles gambiae coreceptor (AgOrco) and other orthologues. These studies reveal that the Orco family can form functional ion channels in the absence of an odor-binding OR, and in addition, demonstrate a first-in-class agonist to further research in insect OR signaling. In light of the extraordinary conservation and widespread expression of the Orco family, VUAA1 represents a powerful new family of compounds that can be used to disrupt the destructive behaviors of nuisance insects, agricultural pests, and disease vectors alike (Jones, 2011).

Other than the unique activity of VUAA1, there are currently no known natural ligands for Orco family members. Therefore, it is suggested that AgOrco and other Orco family members should be recognized as independently gated ion channels or channel subunits rather than ORs (Jones, 2011).

As Orco functionality is required for OR-mediated chemoreception across all insects, an Orco agonist would theoretically be capable of activating all OR-expressing ORNs. Accordingly, Orco agonism would be expected to severely impact the discrimination of odors across all insect taxa, affecting a broad range of chemosensory driven behaviors. In An. gambiae females, universal ORN activation would likely disrupt a variety of olfactory-driven behaviors, most notably human host-seeking, which serves as the foundation for their ability to transmit malaria. The discovery of a universal Orco agonist is also an important step toward the development of a new generation of broad-spectrum agents for integrated management of nuisance insects and agricultural pests (Jones, 2011).

The in vivo VUAA1-mediated activation of AgOrco-expressing cells serves as a proof of principle that targeting AgOrco and other Orco orthologues is a viable approach for the development of behaviorally disruptive olfactory compounds to control a wide range of insect pests and vectors. Although it is premature to speculate as to the ultimate utility of VUAA1 as an antimalarial behaviorally disruptive olfactory compound or as a general modulator of insect chemosensory-driven behaviors, VUAA1 nevertheless represents an important tool for the direct study of AgOrco and other Orco orthologues in insect chemosensory signal transduction (Jones, 2011).

orco mutant mosquitoes lose strong preference for humans and are not repelled by volatile DEET

Female mosquitoes of some species are generalists and will blood-feed on a variety of vertebrate hosts, whereas others display marked host preference. Anopheles gambiae and Aedes aegypti have evolved a strong preference for humans, making them dangerously efficient vectors of malaria and Dengue haemorrhagic fever. Specific host odours probably drive this strong preference because other attractive cues, including body heat and exhaled carbon dioxide (CO2), are common to all warm-blooded hosts. Insects sense odours via several chemosensory receptor families, including the odorant receptors (ORs), membrane proteins that form heteromeric odour-gated ion channels comprising a variable ligand-selective subunit and an obligate co-receptor called Orco. This study used zinc-finger nucleases to generate targeted mutations in the orco gene of A. aegypti to examine the contribution of Orco and the odorant receptor pathway to mosquito host selection and sensitivity to the insect repellent DEET (N,N-diethyl-meta-toluamide). orco mutant olfactory sensory neurons have greatly reduced spontaneous activity and lack odour-evoked responses. Behaviourally, orco mutant mosquitoes have severely reduced attraction to honey, an odour cue related to floral nectar, and do not respond to human scent in the absence of CO2. However, in the presence of CO2, female orco mutant mosquitoes retain strong attraction to both human and animal hosts, but no longer strongly prefer humans. orco mutant females are attracted to human hosts even in the presence of DEET, but are repelled upon contact, indicating that olfactory- and contact-mediated effects of DEET are mechanistically distinct. It is concluded that the odorant receptor pathway is crucial for an anthropophilic vector mosquito to discriminate human from non-human hosts and to be effectively repelled by volatile DEET (DeGennaro, 2013).

Molecular identification and expressive characterization of an olfactory co-receptor gene in the Asian honeybee

Olfaction recognition process is extraordinarily complex in insects, and the olfactory receptors play an important function in the process. This paper describes a highly conserved olfactory co-receptor gene, AcerOr2 (ortholog to the Drosophila melanogaster Or83b), cloned from the antennae of the Asian honeybee, Apis cerana cerana Fabricius (Hymenoptera: Apidae), using reverse transcriptase PCR and rapid amplification of cDNA ends. The full-length sequence of the gene was 1763 bp long, and the cDNA open reading frame encoded 478 amino acid residues, including 7 putative transmembrane domains. Alignment analysis revealed that AcerOr2 shares high homology (> 74%) with similar olfactory receptors found in other Hymenoptera species. The amino acid identity with the closely related species Apis mellifera reached 99.8%. The developmental expression analysis using quantitative real-time reverse transcriptase PCR suggested that the AcerOr2 transcript was expressed at a relatively low level in the larval stage, whereas it was expressed broadly in the pupal and adult stages, with a significantly high level on the days just before and after eclosion. In situ hybridization showed that AcerOr2 mRNA was expressed in sensilla placodea and on the basal region of the worker antennal cuticle, in accordance with the previous conclusions that the conserved genes are expressed in most olfactory receptor neurons (Zhao, 2013).

Molecular Basis of Alarm Pheromone Detection in Aphids

The sesquiterpene (E)-beta-farnesene (EBF) is the alarm pheromone for many species of aphids. When released from aphids attacked by parasitoids or predators, it alerts nearby conspecifics to escape by walking away and dropping off the host plan. The reception of alarm pheromone in aphids is accomplished through a highly sensitive chemosensory system. This study demonstrates that ApisOR5, a member of the large superfamily of odorant receptors, is expressed in large placoid sensillum neurons on the sixth antennal segment and confers response to EBF when co-expressed with Orco, an obligate odorant receptor co-receptor, in parallel heterologous expression systems. In addition, the repellent behavior of Acyrthosiphon pisum to EBF disappears after knocking down ApisOR5 by RNAi as well as two A. pisum odorant-binding proteins known to bind EBF (ApisOBP3 and ApisOBP7). Furthermore, other odorants that can also activate ApisOR5, such as geranyl acetate, significantly repel A. pisum, as does EBF. Taken together, these data lead to the conclusion that ApisOR5 is essential to EBF reception in A. pisum. The characterization of the EBF receptor allows high-throughput screening of aphid repellents, providing the necessary information to develop new strategies for aphid control (Zhang, 2016).

Endogenous insensitivity to the Orco agonist VUAA1 reveals novel olfactory receptor complex properties in the specialist fly Mayetiola destructor

Insect olfactory receptors are routinely expressed in heterologous systems for functional characterisation. It was recently discovered that the essential olfactory receptor co-receptor (Orco) of the Hessian fly, Mayetiola destructor (Mdes), does not respond to the agonist VUAA1, which activates Orco in all other insects analysed to date. Using a mutagenesis-based approach this study identified three residues in MdesOrco, located in different transmembrane helices as supported by 3D modelling, that confer sensitivity to VUAA1. Reciprocal mutations in Drosophila melanogaster (Dmel) and the noctuid moth Agrotis segetum (Aseg) Orcos diminish sensitivity of these proteins to VUAA1. Additionally, mutating these residues in DmelOrco and AsegOrco compromised odourant receptor (OR) dependent ligand-induced Orco activation. In contrast, both wild-type and VUAA1-sensitive MdesOrco were capable of forming functional receptor complexes when coupled to ORs from all three species, suggesting unique complex properties in M. destructor, and that not all olfactory receptor complexes are "created" equal (Corcoran, 2018).


REFERENCES

Search PubMed for articles about Drosophila Orco

Benton, R., Sachse, S., Michnick, S. W. and Vosshall, L. B. (2006). Atypical membrane topology and heteromeric function of Drosophila odorant receptors in vivo. PLoS Biol. 4(2): e20. 16402857

Corcoran, J. A., Sonntag, Y., Andersson, M. N., Johanson, U. and Lofstedt, C. (2018). Endogenous insensitivity to the Orco agonist VUAA1 reveals novel olfactory receptor complex properties in the specialist fly Mayetiola destructor. Sci Rep 8(1): 3489. PubMed ID: 29472565

DasGupta, S. and Waddell, S. (2008). Learned odor discrimination in Drosophila without combinatorial odor maps in the antennal lobe. Curr. Biol. 18(21): 1668-74. PubMed Citation: 18951022

DeGennaro, M., McBride, C. S., Seeholzer, L., Nakagawa, T., Dennis, E. J., Goldman, C., Jasinskiene, N., James, A. A. and Vosshall, L. B. (2013). orco mutant mosquitoes lose strong preference for humans and are not repelled by volatile DEET. Nature 498: 487-491. PubMed ID: 23719379

Del Pino, F., Jara, C., Pino, L. and Godoy-Herrera, R. (2014). The neuro-ecology of Drosophila pupation behavior. PLoS One 9: e102159. PubMed ID: 25033294

Dobritsa, A. A., van der Goes van Naters, W., Warr, C. G., Steinbrecht, R. A. and Carlson, J. R. (2003). Integrating the molecular and cellular basis of odor coding in the Drosophila antenna. Neuron 37: 827-841. 12628173

Elmore, T., Ignell, R., Carlson, J. R. and Smith, D. P. (2003). Targeted mutation of a Drosophila odor receptor defines receptor requirement in a novel class of sensillum. J. Neurosci. 23: 9906-9912. 14586020

Fishilevich, E., Domingos, A. I., Asahina, K., Naef, F., Vosshall, L. B. and Louis, M. (2005). Chemotaxis behavior mediated by single larval olfactory neurons in Drosophila. Curr. Biol. 15(23): 2086-96. 16332533

Getahun, M. N., Wicher, D., Hansson, B. S. and Olsson, S. B. (2012). Temporal response dynamics of Drosophila olfactory sensory neurons depends on receptor type and response polarity. Front Cell Neurosci 6: 54. PubMed ID: 23162431

Getahun, M. N., Olsson, S. B., Lavista-Llanos, S., Hansson, B. S. and Wicher, D. (2013). Insect odorant response sensitivity is tuned by metabotropically autoregulated olfactory receptors. PLoS One 8: e58889. PubMed ID: 23554952

Hallem, E. A., Ho, M. G. and Carlson, J. R. (2004a). The molecular basis of odor coding in the Drosophila antenna. Cell 117: 965-979. 15210116

Hallem, E. A., Fox, A. N., Zwiebel, L. J. and Carlson, J. R. (2004b). Olfaction: mosquito receptor for human-sweat odorant. Nature 427: 212-213. 14724626

Hill, C. A., et al. (2002). G protein-coupled receptors in Anopheles gambiae. Science 298(5591): 176-8. 12364795

Ishii, T., Hirota, J. and Mombaerts, P. (2003). Combinatorial coexpression of neural and immune multigene families in mouse vomeronasal sensory neurons. Curr. Biol. 13: 394-400. 12620187

Jones, P. L., Pask, G. M., Rinker, D. C. and Zwiebel, L. J. (2011). Functional agonism of insect odorant receptor ion channels. Proc Natl Acad Sci U S A 108: 8821-8825. PubMed ID: 21555561

Krieger, J., Klink, O., Mohl, C., Raming, K. and Breer, H. (2003). A candidate olfactory receptor subtype highly conserved across different insect orders. J. Comp. Physiol. [A] 189: 519-526. 12827420

Kwon, Y., et al. (2010). Drosophila TRPA1 channel is required to avoid the naturally occurring insect repellent citronellal. Curr. Biol. 20: 1672-1678. PubMed Citation: 20797863

Larsson, M. C., Domingos, A. I., Jones, W. D., Chiappe, M. E., Amrein, H. and Vosshall, L. B. (2004). Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron 43(5): 703-14. 15339651

Loconto, J., Papes, F., Chang, E., Stowers, L., Jones, E.P., Takada, T., Kumanovics, A., Fischer Lindahl, K. and Dulac, C. (2003). Functional expression of murine V2R pheromone receptors involves selective association with the M10 and M1 families of MHC class molecules. Cell 112: 607-618. 12628182

Mukunda, L., Lavista-Llanos, S., Hansson, B. S. and Wicher, D. (2014). Dimerisation of the Drosophila odorant coreceptor Orco. Front Cell Neurosci 8: 261. PubMed ID: 25221476

Neuhaus, E. M., et al. (2005). Odorant receptor heterodimerization in the olfactory system of Drosophila melanogaster. Nat. Neurosci. 8(1): 15-7. Medline abstract: 15592462

Pitts, R. J., Fox, A. N. and Zwiebel, L. J. (2004). A highly conserved candidate chemoreceptor expressed in both olfactory and gustatory tissues in the malaria vector Anopheles gambiae. Proc. Natl. Acad. Sci. USA 101: 5058-5063. 15037749

Saito, H., Kubota, M., Roberts, R. W., Chi, Q. and Matsunami, H. (2004). RTP family members induce functional expression of mammalian odorant receptors. Cell 119: 679-691. 21720521

Sato, K., Pellegrino, M., Nakagawa, T., Nakagawa, T., Vosshall, L. B. and Touhara, K. (2008). Insect olfactory receptors are heteromeric ligand-gated ion channels. Nature 452(7190): 1002-6. PubMed citation: 18408712 &dopt=Abstract">15550249

Turner, R. M., Derryberry, S. L., Kumar, B. N., Brittain, T., Zwiebel, L. J., Newcomb, R. D. and Christie, D. L. (2014). Mutational analysis of cysteine residues of the insect odorant co-receptor (Orco) from Drosophila melanogaster reveals differential effects on agonist- and odorant/tuning receptor-dependent activation. J Biol Chem 289(46):31837-45.. PubMed ID: 25271160

Vosshall, L. B., Amrein, H., Morozov, P. S., Rzhetsky, A. and Axel, R. (1999). A spatial map of olfactory receptor expression in the Drosophila antenna. Cell 96: 725-736. 10089887

Vosshall, L. B., Wong, A. M. and Axel, R. (2000). An olfactory sensory map in the fly brain. Cell 102: 147-159. 10943836

Wang, J. W., Wong, A. M., Flores, J., Vosshall, L. B. and Axel, R. (2003). Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain. Cell 112: 271-282. 12553914

Wasserman, S., Salomon, A. and Frye, M. A. (2013). Drosophila tracks carbon dioxide in flight. Curr Biol 23: 301-306. PubMed ID: 23352695

Wicher, D. et al. (2008). Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels. Nature 452(7190): 1007-11. PubMed citation: 18408711

Zhang, R., Wang, B., Grossi, G., Falabella, P., Liu, Y., Yan, S., Lu, J., Xi, J. and Wang, G. (2016). Molecular Basis of Alarm Pheromone Detection in Aphids. Curr Biol. PubMed ID: 27916525

Zhao, H., et al. (2013). Molecular identification and expressive characterization of an olfactory co-receptor gene in the Asian honeybee, Apis cerana cerana. J. Insect Sci. 13: 80. PubMed ID: 24224665


Biological Overview

date revised: 20 February 2017

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