To sense myriad environmental odors, animals have evolved multiple, large families of divergent olfactory receptors. How and why distinct receptor repertoires and their associated circuits are functionally and anatomically integrated is essentially unknown. We have addressed these questions through comprehensive comparative analysis of the Drosophila olfactory subsystems that express the ionotropic receptors (IRs) and odorant receptors (ORs). We identify ligands for most IR neuron classes, revealing their specificity for select amines and acids, which complements the broader tuning of ORs for esters and alcohols. IR and OR sensory neurons exhibit glomerular convergence in segregated, although interconnected, zones of the primary olfactory center, but these circuits are extensively interdigitated in higher brain regions. Consistently, behavioral responses to odors arise from an interplay between IR-and OR-dependent pathways. We integrate knowledge on the different phylogenetic and developmental properties of these receptors and circuits to propose models for the functional contributions and evolution of these distinct olfactory subsystems.

No abstract

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 (FRU(M)). However, the volatile pheromone ligands, receptors and olfactory sensory neurons (OSNs) that promote male courtship have not been identified in this important model organism. Here we describe 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 FRU(M)-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 fru(M) courtship circuitry in fruitflies. These findings reveal an unusual but effective evolutionary solution to coordinate feeding and oviposition site selection with reproductive behaviours through a specific sensory pathway.

We hypothesized that cystine/glutamate transporters (xCTs) might be critical regulators of ambient extracellular glutamate levels in the nervous system and that misregulation of this glutamate pool might have important neurophysiological and/or behavioral consequences. To test this idea, we identified and functionally characterized a novel Drosophila xCT gene, which we subsequently named "genderblind" ( gb). Genderblind is expressed in a previously overlooked subset of peripheral and central glia.

The evolution of communication is a fundamental biological problem. The genetic control of the signal and its reception must be tightly coadapted, especially in inter-individual sexual communication. However, there is very little experimental evidence for tight genetic linkage connecting the emission of a signal and its reception. We found that a single genomic transposon inserted in the desat1 gene of Drosophila melanogaster simultaneously affected the emission and the perception of sex-specific signals. This mutation greatly decreased the production of unsaturated hydrocarbons on the cuticle of mature flies of both sexes. These substances represent the sex pheromones necessary for mate discrimination: control males could not discriminate the sex of mutant desat1 flies. Moreover, mutant males were unable to discriminate the sex pheromones of control flies. Expression of desat1 was found in the peripheral tissues that produce and detect sex pheromones. Excision of the transposon rescued both the production and discrimination phenotypes, but the two effects did not always coincide. This indicates that the emission and perception of pheromones are coded by different products of the same gene, reflecting the pleiotropic activity of desat1.

Mate choice is an evolutionarily critical decision that requires the detection of multiple sex-specific signals followed by central integration of these signals to direct appropriate behavior. The mechanisms controlling mate choice remain poorly understood. Here, we show that the glial amino-acid transporter genderblind controls whether Drosophila melanogaster males will attempt to mate with other males. Genderblind (gb) mutant males showed no alteration in heterosexual courtship or copulation, but were attracted to normally unappealing male species-specific chemosensory cues. As a result, genderblind mutant males courted and attempted to copulate with other Drosophila males. This homosexual behavior could be induced within hours using inducible RNAi, suggesting that genderblind controls nervous system function rather than its development. Consistent with this, and indicating that glial genderblind regulates ambient extracellular glutamate to suppress glutamatergic synapse strength in vivo, homosexual behavior could be turned on and off by altering glutamatergic transmission pharmacologically and/or genetically.

One of the most widely used transgenic animal models in biology is Drosophila melanogaster, the fruit fly. Chemical information from this exceedingly small organism is usually accomplished by studying populations to attain sample volumes suitable for standard analysis methods. This paper describes a direct sampling technique capable of obtaining 50-300 nL of hemolymph from individual Drosophila larvae. Hemolymph sampling performed under mineral oil and in air at 30 s intervals up to 120 s after piercing larvae revealed that the effect of evaporation on amino acid concentrations is insignificant when the sample was collected within 60 s. Qualitative and quantitative amino acid analyses of obtained hemolymph were carried out in two optimized buffer conditions by capillary electrophoresis with laser-induced fluorescence detection after derivatizing with fluorescamine. Thirteen amino acids were identified from individual hemolymph samples of both wild-type (WT) control and the genderblind (gb) mutant larvae. The levels of glutamine, glutamate, and taurine in the gb hemolymph were significantly lower at 35%, 38%, and 57% of WT levels, respectively. The developed technique that samples only the hemolymph fluid is efficient and enables accurate organism-level chemical information while minimizing errors associated with possible sample contaminations, estimations, and effects of evaporation compared to the traditional hemolymph-sampling techniques.

SummaryDietary leucine has been suspected to play an important role in insulin release, a hormone that controls satiety and metabolism. The mechanism by which insulin-producing cells (IPCs) sense leucine and regulate insulin secretion is still poorly understood. In Drosophila, insulin-like peptides (DILP2 and DILP5) are produced by brain IPCs and are released in the hemolymph after leucine ingestion. Using Ca2+-imaging and ex vivo cultured larval brains, we demonstrate that IPCs can directly sense extracellular leucine levels via minidiscs (MND), a leucine transporter. MND knockdown in IPCs abolished leucine-dependent changes, including loss of DILP2 and DILP5 in IPC bodies, consistent with the idea that MND is necessary for leucine-dependent DILP release. This, in turn, leads to a strong increase in hemolymph sugar levels and reduced growth. GDH knockdown in IPCs also reduced leucine-dependent DILP release, suggesting that nutrient sensing is coupled to the glutamate dehydrogenase pathway.