InteractiveFly: GeneBrief

Ionotropic receptor 84a: Biological Overview | References


Gene name - Ionotropic receptor 84a

Synonyms -

Cytological map position - 84D6-84D6

Function - channel

Keywords - chemosensory ionotropic glutamate receptor family protein, ion channel, regulation of male courtship behavior, response to aromatic odors

Symbol - Ir84a

FlyBase ID: FBgn0037501

Genetic map position - chr3R:3232504-3235106

Classification - Ligand-gated ion channel

Cellular location - surface transmembrane



NCBI links: HomoloGene | EntrezGene
BIOLOGICAL OVERVIEW

Many animals attract mating partners through the release of volatile sex pheromones, which can convey information on the species, gender and receptivity of the sender to induce innate courtship and mating behaviours by the receiver. Male Drosophila melanogaster fruitflies display stereotyped reproductive behaviours towards females, and these behaviours are controlled by the neural circuitry expressing male-specific isoforms of the transcription factor Fruitless (FRUM). However, the volatile pheromone ligands, receptors and olfactory sensory neurons (OSNs) that promote male courtship have not been identified in this important model organism. This study describes a novel courtship function of Ionotropic receptor 84a (IR84a), which is a member of the chemosensory ionotropic glutamate receptor family, in a previously uncharacterized population of FRUM-positive OSNs. IR84a-expressing neurons are activated not by fly-derived chemicals but by the aromatic odours phenylacetic acid and phenylacetaldehyde, which are widely found in fruit and other plant tissues that serve as food sources and oviposition sites for drosophilid flies. Mutation of Ir84a abolishes both odour-evoked and spontaneous electrophysiological activity in these neurons and markedly reduces male courtship behaviour. Conversely, male courtship is increased -- in an IR84a-dependent manner -- in the presence of phenylacetic acid but not in the presence of another fruit odour that does not activate IR84a. Interneurons downstream of IR84a-expressing OSNs innervate a pheromone-processing centre in the brain. Whereas IR84a orthologues and phenylacetic-acid-responsive neurons are present in diverse drosophilid species, IR84a is absent from insects that rely on long-range sex pheromones. Our results suggest a model in which IR84a couples food presence to the activation of the fruM courtship circuitry in fruit flies. These findings reveal an unusual but effective evolutionary solution to coordinate feeding and oviposition site selection with reproductive behaviours through a specific sensory pathway (Grosjean, 2012).

While mapping the projections of Ionotropic receptor (IR)-expressing OSNs to the primary olfactory centre (Silbering, 2011), the antennal lobe, it was observed that an Ir84a reporter was labelling neurons innervating the VL2a glomerulus. VL2a is one of only three glomeruli that are larger in males and whose OSN inputs and projection neuron outputs express male-specific isoforms of the behavioural sex determination gene fruitless (fruM) (Stockinger, 2005). fruM-expressing OSNs have been implicated in promoting male sexual behaviours, because inhibition of synaptic transmission in all of these neurons simultaneously reduces male courtship of female. The expression of Ir84a in fruM-expressing neurons was confirmed by visualizing the co-expression of an Ir84a reporter, as well as endogenous Ir84a transcripts, with a fruM reporter. No sexual dimorphism was observed either in the number of Ir84a-expressing cells or in their targeting to VL2a, indicating that FRUM does not have an essential role in the development of these neurons, similar to other fruM-expressing OSNs (Grosjean, 2012).

A GAL4 knock-in null allele, Ir84aGAL4 was generated. Ir84aGAL4/+ heterozygotes expressed a GAL4-responsive, membrane-targeted, green fluorescent protein (GFP) transgene (UAS-mCD8:GFP) exclusively in Ir84a-expressing OSNs. In Ir84aGAL4 homozygotes, the endogenous expression of Ir84a was lost, but the distribution and dendritic projections of these neurons, as revealed by mCD8:GFP, was unaffected. The axons of Ir84a-expressing neurons in heterozygous and homozygous Ir84aGAL4 flies projected only to VL2a. Ir84a is therefore dispensable for the specification and wiring of the neurons in which it is expressed. An amino-terminal enhanced GFP (EGFP)-tagged version of this receptor (Abuin, 2011) localized to the cell bodies and the ciliated dendritic endings of these neurons but not to their axon termini, consistent with an exclusive role for IR84a as an olfactory receptor in the fruM circuitry (Grosjean, 2012).

The responses of IR84a-expressing neurons to chemicals produced by male or virgin female flies was tested, both by delivering headspaces of flies from a distance (simulating the action of volatile pheromones) and by presenting extracts from fly cuticles at close range (mimicking exposure to non-volatile hydrocarbons, such as contact pheromones. These stimuli produced no or extremely small responses, as detected by extracellular recordings in ac4 sensilla, which belong to the class of olfactory hair that houses IR84a-expressing neurons, as well as OSNs that express IR75d, or IR76a and IR76b (Benton, 2009). These observations suggest that IR84a is not tuned to fly-derived pheromones. Therefore 163 structurally diverse odours were tested. Only three of these gave responses of >50 spikes s−1 above basal activity: phenylacetaldehyde [as identified previously (Yao, 2005)], phenylacetic acid and phenylethylamine. Dose response curves that revealed sensitivity to these ligands are similar in both sexes (Grosjean, 2012).

In Ir84aGAL4 homozygous mutants, the responses to phenylacetic acid and phenylacetaldehyde were completely abolished. Re-introduction of Ir84a function in these neurons, by using UAS-Ir84a or UAS-EGFP:Ir84a cDNA transgenes, rescued these phenotypes, indicating a cell-autonomous function for IR84a in mediating these odour responses. By contrast, responses to phenylethylamine were unaffected, corroborating the evidence that this chemical is detected by the neurons that express both IR76a and IR76b. Consistent with these loss-of-function data, misexpression of IR84a in Odorant receptor 35a (OR35a)-expressing neurons was sufficient to confer responsiveness to phenylacetic acid and phenylacetaldehyde. The basal activity in Ir84a mutant ac4 sensilla was also lower than that in the ac4 sensilla of wild-type and rescue genotypes, indicating that IR84a has a role in promoting spontaneous firing (Grosjean, 2012).

Phenylacetic acid and phenylacetaldehyde are aromatic compounds found in a diverse range of fruit and other plant tissues, as well as in their fermentation products, and they are used in human perfumes for their floral, honey-like, sweet smell. The presence of these chemicals in two host fruit for drosophilid flies, overripe bananas and the prickly-pear cactus Opuntia ficus-indica, as well as in laboratory Drosophila medium, was confirmed by using gas chromatography- mass spectrometry analysis. The ubiquity of phenylacetic acid in vegetal tissues may be linked with its activity as a growth-regulating auxin and/or its production by plant-associated microorganisms. Small, but reproducible, quantities of phenylacetic acid and phenylacetaldehyde were also detected in whole-body cuticular extracts of male and virgin female D. melanogaster. The similarity in the relative amounts of these chemicals in laboratory medium and fruitfly extracts suggested that these chemicals are transferred from food to flies during their culture. 'Clean' cuticular extracts from animals grown on a minimal medium containing only sucrose and agarose consistently contain no detectable phenylacetaldehyde or phenylacetic acid (Grosjean, 2012).

The expression of IR84a in fruM-expressing neurons implicates this receptor in the regulation of male courtship (Manoli, 2005; Stockinger, 2005). Indeed, in single-pair courtship assays, Ir84aGAL4 mutant males court wild-type females significantly less than do wild-type males. This phenotype was observed using both decapitated virgin females (which do not produce feedback signals) and in more natural conditions, with intact females together with food. Most individual components of the courtship ritual were affected in Ir84a mutant flies. These defects were rescued with a UAS-Ir84a transgene, confirming that they result from the absence of IR84a in OSNs. The observed reduction in male heterosexual courtship index (~50%) is highly comparable to the phenotype of flies in which all FRUM-positive OSNs are silenced (Stockinger, (2005), suggesting that IR84a-expressing neurons are the major olfactory fruM channel contributing to this behaviour. Residual courtship is presumably stimulated by other sensory modalities, such as taste. Male wild-type D. melanogaster also show a low level of courtship towards other males, and this homosexual courtship was also markedly reduced in Ir84aGAL4 mutants. By contrast, Ir84aGAL4 mutant females did not show overt defects in reproductive behaviours, including copulation latency, success or duration (Grosjean, 2012).

In innate olfactory preference assays, Ir84aGAL4 mutant flies still show robust avoidance of acetic acid, indicating that they do not have a general impairment in sensory detection. By contrast, no obvious responses of flies to phenylacetic acid was observed, suggesting that this food-derived odour is not a volatile stimulus that attracts flies but is a salient cue at close range. Notably, phenylacetic acid has a low vapour pressure compared with other fruit volatiles (for example, ethyl butyrate). The observation that courtship is reduced in Ir84aGAL4 mutants in assays in which only small amounts of phenylacetic acid are present on fly cuticles raises the possibility that spontaneous activity of these neurons also contributes to establishing a basal courtship level, which is abolished in the absence of IR84a (Grosjean, 2012).

To test whether IR84a ligands are sufficient to promote courtship, the assay was adapted by using killed female objects (which males court at only low levels) and by replacing the base of the chamber with gauze, beneath which a filter paper treated with odour or solvent was placed. Perfuming with phenylacetic acid nearly doubled the courtship index of wild-type flies compared with a solvent control. This effect was abolished in Ir84aGAL4 mutants and could be restored, albeit not fully, by introducing a UAS-Ir84a transgene. By contrast, ethyl butyrate, which does not activate IR84a, did not increase courtship. The courtship chamber was also perfumed with Drosophila food—which contains phenylacetic acid, and this complex olfactory stimulus was observed to induced IR84a-dependent increases in male courtship behaviour (Grosjean, 2012).

The other fruM-expressing OSN populations express either OR67d, which is a receptor for the antiaphrodisiac male pheromone cis-vaccenyl acetate, or OR47b, which is activated by unidentified fly-derived odours from both sexes and may participate in mate localization. How IR84a sensory information is integrated with these pheromonal pathways was examined by visualizing the axons of projection neurons innervating the VL2a (IR84a), VA1lm (OR47b) and DA1 (OR67d) glomeruli, which carry sensory information to the mushroom body and lateral horn. Images of single-labelled projection neurons of different glomerular classes were registered onto a common reference brain. DA1 and VA1lm excitatory projection neurons target an anterior-ventral pheromone-processing region of the lateral horn, which is segregated from projection neurons that are responsive to general food odours. Importantly, it was found that VL2a projection neurons—and no other IR-expressing projection neuron class are highly interdigitated with pheromone pathways and not food pathways. Indeed, VL2a projection neuron axon terminals overlap more strongly with VA1lm projection neurons than any of the other 44 projection neuron classes, consistent with projection neurons of both of these classes transmitting courtship-promoting sensory signals. The VL2a, DA1 and VA1lm inhibitory projection neurons were observed to overlap to a similar extent. The anatomical convergence of combinations of excitatory and inhibitory inputs from VL2a, VA1lm and DA1 projection neurons may allow the integration of olfactory signals by fruM-expressing third-order neurons (Cachero, 2010; Yu, 2010) to control male courtship behaviour (Grosjean, 2012).

Many olfactory IRs are conserved in insects (Croset, 2010) and may detect odours that are important for all species. By contrast, although IR84a orthologues are present in ecologically diverse drosophilids, they are absent from other Diptera and more divergent insects. In the cactophilic species Drosophila mojavensis, coeloconic sensilla with neurons were identified that are responsive to phenylacetic acid and phenylacetaldehyde on their anterior antennal surface (similar to ac4 sensilla in D. melanogaster). Thus, IR84a may have a conserved, drosophilid-specific function (Grosjean, 2012).

Despite the widely held assumption of the existence of volatile chemicals that promote courtship in Drosophila, behavioural evidence for long-range pheromones is inconclusive, and no female-specific volatile compound that activates male OSNs has been identified. The characterization of IR84a identifies an olfactory receptor that is expressed in FRUM-positive neurons and is required to promote male courtship. Surprisingly, this receptor is not activated by fly-derived odours but rather by aromatic compounds that are present in the vegetal substrates in which fruitflies feed, breed and oviposit. Thus, the IR84a pathway may promote male courtship in the presence of food, complementing the functions of pheromone receptors in regulating mate choice. This model can account for the widespread observations that D. melanogaster and other drosophilids mate predominantly on their food substrates. Whereas many insects and other animal classes use long-range sex pheromones to attract potential mates, the evolution of IR84a in fruitflies has provided an alternative (although not necessarily exclusive) olfactory mechanism to unite males with females by integrating food-sensing neurons with the circuitry controlling sexual behaviour. Whether other animals have dedicated sensory pathways for environmental 'aphrodisiacs' remains an open question (Grosjean, 2012).


REFERENCES

Search PubMed for articles about Drosophila Ir84a

Abuin, L., Bargeton, B., Ulbrich, M. H., Isacoff, E. Y., Kellenberger, S. and Benton, R. (2011). Functional architecture of olfactory ionotropic glutamate receptors. Neuron 69: 44-60. PubMed ID: 21220098

Benton, R., Vannice, K. S., Gomez-Diaz, C. and Vosshall, L. B. (2009). Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell 136: 149-162. PubMed ID: 19135896

Cachero, S., Ostrovsky, A. D., Yu, J. Y., Dickson, B. J. and Jefferis, G. S. (2010). Sexual dimorphism in the fly brain. Curr Biol 20: 1589-1601. PubMed ID: 20832311

Croset, V., Rytz, R., Cummins, S. F., Budd, A., Brawand, D., Kaessmann, H., Gibson, T. J. and Benton, R. (2010). Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction. PLoS Genet 6: e1001064. PubMed ID: 20808886

Grosjean, Y., Rytz, R., Farine, J. P., Abuin, L., Cortot, J., Jefferis, G. S. and Benton, R. (2011). An olfactory receptor for food-derived odours promotes male courtship in Drosophila. Nature 478: 236-240. PubMed ID: 21964331

Manoli, D. S., Foss, M., Villella, A., Taylor, B. J., Hall, J. C. and Baker, B. S. (2005). Male-specific fruitless specifies the neural substrates of Drosophila courtship behaviour. Nature 436: 395-400. PubMed ID: 15959468

Silbering, A. F., Rytz, R., Grosjean, Y., Abuin, L., Ramdya, P., Jefferis, G. S. and Benton, R. (2011). Complementary function and integrated wiring of the evolutionarily distinct Drosophila olfactory subsystems. J Neurosci 31: 13357-13375. PubMed ID: 21940430

Stockinger, P., Kvitsiani, D., Rotkopf, S., Tirian, L. and Dickson, B. J. (2005). Neural circuitry that governs Drosophila male courtship behavior. Cell 121: 795-807. PubMed ID: 15935765

Yao, C. A., Ignell, R. and Carlson, J. R. (2005). Chemosensory coding by neurons in the coeloconic sensilla of the Drosophila antenna. J Neurosci 25: 8359-8367. PubMed ID: 16162917

Yu, J. Y., Kanai, M. I., Demir, E., Jefferis, G. S. and Dickson, B. J. (2010). Cellular organization of the neural circuit that drives Drosophila courtship behavior. Curr Biol 20: 1602-1614. PubMed ID: 20832315


Biological Overview

date revised: 6 January 2013

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