Summary Plants produce naturally occurring insect repellents, such as citronellal, which is the main component of citronellal oil and is among the most widely-used-naturally-occurring insect repellents. However, the molecular pathways through which insects sense botanical repellents are unknown. Here, we showed that Drosophila used two pathways for direct avoidance of citronellal. The olfactory co-receptor, Or83b, which is required for the response to the synthetic repellent DEET, contributed to citronellal repulsion, and was essential for citronellal-evoked action potentials. Mutations affecting the Ca2+-permeable cation channel, TRPA1 resulted in a comparable defect in avoiding citronellal vapor. The TRPA1-dependent aversion to citronellal relied on a G protein/phospholipase C (PLC) signaling cascade, rather than direct detection of citronellal by TRPA1. Loss of TRPA1, Gq or PLC caused an increase in the frequency of citronellal-evoked action potentials in olfactory receptor neurons. Absence of the Ca2+-activated K+ channel, Slowpoke, resulted 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 was 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.

We set out to deorphanize a subset of putative Drosophila odorant receptors expressed in trichoid sensilla using a transgenic in vivo misexpression approach. We identified farnesol as a potent and specific activator for the orphan odorant receptor Or83c. Farnesol is an intermediate in juvenile hormone biosynthesis, but is also produced by ripe citrus fruit peels. Here, we show that farnesol stimulates robust activation of Or83c-expressing olfactory neurons, even at high dilutions. The CD36 homolog Snmp1 is required for normal farnesol response kinetics. The neurons expressing Or83c are found in a subset of poorly characterized intermediate sensilla. We show that these neurons mediate attraction behavior to low concentrations of farnesol and that Or83c receptor mutants are defective for this behavior. Or83c neurons innervate the DC3 glomerulus in the antennal lobe and projection neurons relaying information from this glomerulus to higher brain centers target a region of the lateral horn previously implicated in pheromone perception. Our findings identify a sensitive, narrowly tuned receptor that mediates attraction behavior to farnesol and demonstrates an effective approach to deorphanizing odorant receptors expressed in neurons located in intermediate and trichoid sensilla that may not function in the classical "empty basiconic neuron" system.

The molecular and cellular events mediating complex behaviors in animals are largely unknown. Elucidating the circuits underlying behaviors in simple model systems may shed light on how these circuits function. In Drosophila, courtship behavior provides a tractable model for studying the underlying basis of innate behavior. The male-specific pheromone 11-cis-vaccenyl acetate (cVA) modulates courtship behavior and is detected by T1 neurons, located on the antenna of male and female flies. The T1 neurons express the odorant receptor Or67d, and are exquisitely tuned to cVA pheromone. However, cVA-induced changes in mating behavior have also been reported upon manipulation of olfactory neurons expressing odorant receptor Or65a. These findings raise the issue of whether multiple olfactory-driven circuits underlie cVA-induced behavioral responses, and what role these circuits play in behavior. Here, we engineered flies in which the Or67d circuit is specifically activated in the absence of cVA in order to determine the role of this circuit in behavior. We created transgenic flies that express a dominant-active, pheromone-independent variant of the extracellular pheromone receptor, LUSH. We found that, similar to the behaviors elicited by cVA, engineered male flies have dramatically reduced courtship, while engineered females showed enhanced courtship. Furthermore, cVA exposure did not enhance the dominant LUSH-triggered effects on behavior in the engineered flies. Finally, we show the effects of both cVA and dominant LUSH on courtship are reversed by genetically removing Or67d. These findings demonstrate that the T1/Or67d circuit is necessary and sufficient to mediate sexually dimorphic courtship behaviors.

Insects have extraordinary sensitivity to volatile odorants. In the case of pheromone reception, sensitivity approaches the single molecule detection limit. The mystery of how insects detect volatile odorants has intrigued researchers for more than a century, but only recently have the molecular underpinnings been uncovered. In this review we summarize recent work that reveals multiple signaling mechanisms utilizing several distinct receptor families underlie olfaction in Drosophila. Some of these mechanisms have not been observed in other animals, suggesting they evolved relatively late along an evolutionary branch that ultimately gave rise to the insects.