Drosophila melanogaster olfactory neurons have long been thought to express only one chemosensory receptor gene family. There are two main olfactory receptor gene families in Drosophila, the odorant receptors (ORs) and the ionotropic receptors (IRs). The dozens of odorant-binding receptors in each family require at least one co-receptor gene in order to function: Orco for ORs, and Ir25a, Ir8a, and Ir76b for IRs. Using a new genetic knock-in strategy, we targeted the four co-receptors representing the main chemosensory families in D. melanogaster (Orco, Ir8a, Ir76b, Ir25a). Co-receptor knock-in expression patterns were verified as accurate representations of endogenous expression. We find extensive overlap in expression among the different co-receptors. As defined by innervation into antennal lobe glomeruli, Ir25a is broadly expressed in 88% of all olfactory sensory neuron classes and is co-expressed in 82% of Orco+ neuron classes, including all neuron classes in the maxillary palp. Orco, Ir8a, and Ir76b expression patterns are also more expansive than previously assumed. Single sensillum recordings from Orco-expressing Ir25a mutant antennal and palpal neurons identify changes in olfactory responses. We also find co-expression of Orco and Ir25a in Drosophila sechellia and Anopheles coluzzii olfactory neurons. These results suggest that co-expression of chemosensory receptors is common in insect olfactory neurons. Together, our data present the first comprehensive map of chemosensory co-receptor expression and reveal their unexpected widespread co-expression in the fly olfactory system.

Drosophila olfactory neurons have long been thought to express only one chemosensory receptor modality. Using a new genetic knock-in strategy, we targeted the four co-receptors representing the chemosensory modes in Drosophila (Orco, Ir25a, Ir8a, Ir76b). Co-receptor knock-in expression patterns were verified as accurate representations of endogenous expression. We find extensive overlap in expression among the different co-receptors. As defined by innervation into antennal lobe glomeruli, Ir25a is broadly expressed in 88% of olfactory neuron classes and is co-expressed in 64% of Orco+ neuron classes, including all neuron classes in the maxillary palp. Orco, Ir8a, and Ir76b expression patterns are also expanded. Single sensillum recordings from Orco-expressing Ir25a mutant antennal and palpal neurons identify significant changes in olfactory responses. These results suggest polymodal expression and function of chemosensory receptors is common in olfactory neurons. We present a new map of the olfactory system reflecting this polymodal expression.

Highlights d Ammonium transporters label previously uncharacterized neuron populations d These olfactory neurons selectively respond to ammonia d The Amt transporter acts as a non-canonical olfactory receptor in Drosophila d The function of Amt may be conserved across insect species

Two large families of olfactory receptors, the Odorant Receptors (ORs) and Ionotropic Receptors (IRs), mediate responses to most odors in the insect olfactory system. Individual odorant binding “tuning” OrX receptors are expressed by olfactory neurons in basiconic and trichoid sensilla and require the co-receptor Orco. The situation for IRs is more complex. Different tuning IrX receptors are expressed by olfactory neurons in coeloconic sensilla and rely on either the Ir25a or Ir8a co-receptors; some evidence suggests that Ir76b may also act as a co-receptor, but its function has not been systematically examined. Surprisingly, recent data indicate that nearly all coeloconic olfactory neurons co-express Ir25a, Ir8a, and Ir76b. Here, we demonstrate that Ir76b and Ir25a function together in all amine-sensing olfactory receptor neurons. In most neurons, loss of either co-receptor abolishes amine responses. In contrast, amine responses persist in the absence of Ir76b or Ir25a in ac1 sensilla but are lost in a double mutant. We show that responses mediated by acid-sensing neurons do not require Ir76b, despite their expression of this co-receptor. Our study also demonstrates that one population of coeloconic olfactory neurons exhibits Ir76b/Ir25a-dependent and Orco-dependent responses to distinct odorants. Together, our data establish the role of Ir76b as a bona fide co-receptor, which acts in partnership with Ir25a. Given that these co-receptors are among the most highly conserved olfactory receptors and are often co-expressed in chemosensory neurons, our data suggest Ir76b and Ir25a also work in tandem in other insects.

Two families of ligand-gated ion channels function as olfactory receptors in insects. Here, we show that these canonical olfactory receptors are not necessary for responses to ammonia, a key ecological odor that is attractive to many insects including disease vectors and agricultural pests. Instead, we show that a member of the ancient electrogenic ammonium transporter family, Amt, is a new type of olfactory receptor. We report two hitherto unidentified olfactory neuron populations that mediate neuronal and behavioral responses to ammonia. Their endogenous ammonia responses are Amt-dependent, and ectopic expression of either Drosophila or Anopheles Amt confers ammonia sensitivity. Amt is the first transporter known to function as an olfactory receptor in animals, and its role may be conserved across insect species.