Structural and functional differences in the antennal olfactory system of worker honey bees of Apis mellifera and Apis cerana

Olfactory cues are important sensory modalities on individual discrimination, perception, and efficient orientation to food sources in most insects. In honey bees, which are well known as eusocial insects, olfactory cues are mainly used to maintain a colony. Although much research has been reported on olfactory systems in honey bee olfaction, little is known about the differences between two major honey bee species, the European honey bee Apis mellifera and the Asian honey bee Apis cerana. In order to understand the differences of olfactory characteristics in the two species, we compared the distribution of sensory hairs on the antennae and antennal olfactory responses, using electron microscopy, electrophysiological recording and molecular expression level of odorant receptors. Our present study demonstrated that the antennae of A. cerana have more olfactory sensilla than A. mellifera, responding more strongly to various floral volatile compounds. At the molecular level, olfactory co-receptor (Orco), which makes heterodimers with other conventional olfactory receptors, is more abundantly expressed in the antenna of A. cerana than of A. mellifera. These findings extend our understanding of the olfactory systems and behavioral responses to various ecological and biological signals in two closely related honey bee species.     

Two molecular motility systems of the frog olfactory cilia

Intravital video microscopy was used to study the motility of frog (Rana temporaria) olfactory cilia exposed to various odorants—pentanol, camphor, cineole, and vanillin (first group); ammonia and hydrogen sulfide (second group)—and to the cell respiration inhibitors rotenone and malonate. It was demonstrated that the olfactory cilia had both the dynein-tubulin and actin-myosin molecular motility systems, the former providing unordered and the latter, ordered movements. The motility became ordered in response to exposure to odorants. The tested odorants belonging to different groups had different effects on the mitochondrial respiratory chain activity and the motility of olfactory cilia.     

Advantage of the Highly Restricted Odorant Receptor Expression Pattern in Chemosensory Neurons of Drosophila

A fundamental molecular feature of olfactory systems is that individual neurons express only one receptor from a large odorant receptor gene family. While numerous theories have been proposed, the functional significance and evolutionary advantage of generating a sophisticated one-receptor-per neuron expression pattern is not well understood. Using the genetically tractable Drosophila melanogaster as a model, we demonstrate that the breakdown of this highly restricted expression pattern of an odorant receptor in neurons leads to a deficit in the ability to exploit new food sources. We show that animals with ectopic co-expression of odorant receptors also have a competitive disadvantage in a complex environment with limiting food sources. At the level of the olfactory system, we find changes in both the behavioral and electrophysiological responses to odorants that are detected by endogenous receptors when an olfactory receptor is broadly misexpressed in chemosensory neurons. Taken together these results indicate that restrictive expression patterns and segregation of odorant receptors to individual neuron classes are important for sensitive odor-detection and appropriate olfactory behaviors.     

Chemosensory apparatus of Drosophila larvae

Many insects, including Drosophila melanogaster, have a rich repertoire of olfactory behavior. Combination of robust behavioral assays, physiological and molecular tools render D. melanogaster as highly suitable system for olfactory studies. The small number of neurons in the olfactory system of fruit flies, especially the number of sensory neurons in the larval stage, makes the exploration of sensory coding at all stages of its nervous system a potentially tractable goal, which is not possible in the foreseeable future in any mammalian preparation. Advances in physiological recordings, olfactory signaling and detailed analysis of behavior, can place larvae in a position to ask previously unanswerable questions.     

Molecular determinants of odorant receptor function in insects

The olfactory system of Drosophila melanogaster provides a powerful model to study molecular and cellular mechanisms underlying function of a sensory system. In the 1970s Siddiqi and colleagues pioneered the application of genetics to olfactory research and isolated several mutant Drosophila with odorant-specific defects in olfactory behaviour, suggesting that odorants are detected differentially by the olfactory system. Since then basic principles of olfactory system function and development have emerged using Drosophila as a model. Nearly four decades later we can add computational methods to further our understanding of how specific odorants are detected by receptors. Using a comparative approach we identify two categories of short amino acid sequence motifs: ones that are conserved family-wide predominantly in the C-terminal half of most receptors, and ones that are present in receptors that detect a specific odorant, 4-methylphenol, found predominantly in the N-terminal half. The odorant-specific sequence motifs are predictors of phenol detection in Anopheles gambiae and other insects, suggesting they are likely to participate in odorant binding. Conversely, the family-wide motifs are expected to participate in shared functions across all receptors and a mutation in the most conserved motif leads to a reduction in odor response. These findings lay a foundation for investigating functional domains within odorant receptors that can lead to a molecular understanding of odor detection.     

Molecular Clock Regulates Daily α1–2-Fucosylation of the Neural Cell Adhesion Molecule (NCAM) within Mouse Secondary Olfactory Neurons

The circadian clock regulates various behavioral and physiological rhythms in mammals. Circadian changes in olfactory functions such as neuronal firing in the olfactory bulb (OB) and olfactory sensitivity have recently been identified, although the underlying molecular mechanisms remain unknown. We analyzed the temporal profiles of glycan structures in the mouse OB using a high-density microarray that includes 96 lectins, because glycoconjugates play important roles in the nervous system such as neurite outgrowth and synaptogenesis. Sixteen lectin signals significantly fluctuated in the OB, and the intensity of all three that had high affinity for α1–2-fucose (α1–2Fuc) glycan in the microarray was higher during the nighttime. Histochemical analysis revealed that α1–2Fuc glycan is located in a diurnal manner in the lateral olfactory tract that comprises axon bundles of secondary olfactory neurons. The amount of α1–2Fuc glycan associated with the major target glycoprotein neural cell adhesion molecule (NCAM) varied in a diurnal fashion, although the mRNA and protein expression of Ncam1 did not. The mRNA and protein expression of Fut1, a α1–2-specific fucosyltransferase gene, was diurnal in the OB. Daily fluctuation of the α1–2Fuc glycan was obviously damped in homozygous Clock mutant mice with disrupted diurnal Fut1 expression, suggesting that the molecular clock governs rhythmic α1–2-fucosylation in secondary olfactory neurons. These findings suggest the possibility that the molecular clock is involved in the diurnal regulation of olfaction via α1–2-fucosylation in the olfactory system.     

Cloning and expression pattern of a putative octopamine/tyramine receptor in antennae of the noctuid moth <Emphasis Type="Italic">Mamestra brassicae</Emphasis>

In insects, biogenic amines have been shown to play an important role in olfactory plasticity. In a first attempt to decipher the underlying molecular mechanisms, we report the molecular cloning and precise expression pattern of a newly identified octopamine/tyramine-receptor-encoding gene in the antennae of the noctuid moth Mamestra brassicae (MbraOAR/TAR). A full-length cDNA has been obtained through homology cloning in combination with rapid amplification of cDNA ends/polymerase chain reaction; the deduced protein exhibits high identities with previously identified octopamine/tyramine receptors in other moths. In situ hybridization within the antennae has revealed that MbraOAR/TAR is expressed at the bases of both pheromone-sensitive and non-sensitive olfactory sensilla and in cells with a neurone-like shape. In accordance with previous physiological studies that have revealed a role of biogenic amines in the electrical activity of the receptor neurones, our results suggest that biogenic amines (either octopamine or tyramine) target olfactory receptor neurones to modulate olfactory coding as early as the antennal level.     

Phospholipase C and Diacylglycerol Mediate Olfactory Responses to Amino Acids in the Main Olfactory Epithelium of an Amphibian

The semi-aquatic lifestyle of amphibians represents a unique opportunity to study the molecular driving forces involved in the transition of aquatic to terrestrial olfaction in vertebrates. Most amphibians have anatomically segregated main and vomeronasal olfactory systems, but at the cellular and molecular level the segregation differs from that found in mammals. We have recently shown that amino acid responses in the main olfactory epithelium (MOE) of larval Xenopus laevis segregate into a lateral and a medial processing stream, and that the former is part of a vomeronasal type 2 receptor expression zone in the MOE. We hypothesized that the lateral amino acid responses might be mediated via a vomeronasal-like transduction machinery. Here we report that amino acid-responsive receptor neurons in the lateral MOE employ a phospholipase C (PLC) and diacylglycerol-mediated transduction cascade that is independent of Ca²⁺ store depletion. Furthermore, we found that putative transient receptor potential (TRP) channel blockers inhibit most amino acid-evoked responses in the lateral MOE, suggesting that ion channels belonging to the TRP family may be involved in the signaling pathway. Our data show, for the first time, a widespread PLC- and diacylglycerol-dependent transduction cascade in the MOE of a vertebrate already possessing a vomeronasal organ.     

Peripheral regulation by ecdysteroids of olfactory responsiveness in male Egyptian cotton leaf worms, Spodoptera littoralis

Physiological and behavioral plasticity allows animals to adapt to changes in external (environmental) and internal (physiological) factors. In insects, the physiological state modulates adult behavior in response to different odorant stimuli. Hormones have the potential to play a major role in the plasticity of the olfactory responses. To explore if peripheral olfactory processing could be regulated by steroid hormones, we characterized the molecular, electrophysiological, and behavioral response to changes in endogenous hormone levels in adult male Spodoptera littoralis. The expression of the receptor complex (EcR/USP) was localized by in situ hybridization in the olfactory sensilla of antennae. Injections of 20-hydroxyecdysone (20E) induced an ecdysteroid signaling pathway in antennae and increased expression of the nuclear receptors EcR, USP and E75. Diacylglycerol kinase (DGK) and CaM expression were also up-regulated by 20E. Taken together, these molecular, electrophysiological, and behavioral results suggest a hormonal regulation of the peripheral olfactory processing in S. littoralis.     

Conserved roles of ems/Emx and otd/Otx genes in olfactory and visual system development in Drosophila and mouse

The regional specialization of brain function has been well documented in the mouse and fruitfly. The expression of regulatory factors in specific regions of the brain during development suggests that they function to establish or maintain this specialization. Here, we focus on two such factors—the Drosophila cephalic gap genes empty spiracles (ems) and orthodenticle (otd), and their vertebrate homologues Emx1/2 and Otx1/2—and review novel insight into their multiple crucial roles in the formation of complex sensory systems. While the early requirement of these genes in specification of the neuroectoderm has been discussed previously, here we consider more recent studies that elucidate the later functions of these genes in sensory system formation in vertebrates and invertebrates. These new studies show that the ems and Emx genes in both flies and mice are essential for the development of the peripheral and central neurons of their respective olfactory systems. Moreover, they demonstrate that the otd and Otx genes in both flies and mice are essential for the development of the peripheral and central neurons of their respective visual systems. Based on these recent experimental findings, we discuss the possibility that the olfactory and visual systems of flies and mice share a common evolutionary origin, in that the conserved visual and olfactory circuit elements derive from conserved domains of otd/Otx and ems/Emx action in the urbilaterian ancestor.     

The stimulatory Gα(s) protein is involved in olfactory signal transduction in Drosophila

Seven-transmembrane receptors typically mediate olfactory signal transduction by coupling to G-proteins. Although insect odorant receptors have seven transmembrane domains like G-protein coupled receptors, they have an inverted membrane topology, constituting a key difference between the olfactory systems of insects and other animals. While heteromeric insect ORs form ligand-activated non-selective cation channels in recombinant expression systems, the evidence for an involvement of cyclic nucleotides and G-proteins in odor reception is inconsistent. We addressed this question in vivo by analyzing the role of G-proteins in olfactory signaling using electrophysiological recordings. We found that Gα_s plays a crucial role for odorant induced signal transduction in OR83b expressing olfactory sensory neurons, but not in neurons expressing CO₂ responsive proteins GR21a/GR63a. Moreover, signaling of Drosophila ORs involved Gα_s also in a heterologous expression system. In agreement with these observations was the finding that elevated levels of cAMP result in increased firing rates, demonstrating the existence of a cAMP dependent excitatory signaling pathway in the sensory neurons. Together, we provide evidence that Gα_s plays a role in the OR mediated signaling cascade in Drosophila.     

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann^1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor³ for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L''Etoile et al.,⁴ uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC⁴. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC⁵. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC^6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.     

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.     

A screen for genes expressed in the olfactory organs of Drosophila melanogaster identifies genes involved in olfactory behaviour

Background

For insects the sense of smell and associated olfactory-driven behaviours are essential for survival. Insects detect odorants with families of olfactory receptor proteins that are very different to those of mammals, and there are likely to be other unique genes and genetic pathways involved in the function and development of the insect olfactory system.

Methodology/Principal Findings

We have performed a genetic screen of a set of 505 Drosophila melanogaster gene trap insertion lines to identify novel genes expressed in the adult olfactory organs. We identified 16 lines with expression in the olfactory organs, many of which exhibited expression of the trapped genes in olfactory receptor neurons. Phenotypic analysis showed that six of the lines have decreased olfactory responses in a behavioural assay, and for one of these we showed that precise excision of the P element reverts the phenotype to wild type, confirming a role for the trapped gene in olfaction. To confirm the identity of the genes trapped in the lines we performed molecular analysis of some of the insertion sites. While for many lines the reported insertion sites were correct, we also demonstrated that for a number of lines the reported location of the element was incorrect, and in three lines there were in fact two pGT element insertions.

Conclusions/Significance

We identified 16 new genes expressed in the Drosophila olfactory organs, the majority in neurons, and for several of the gene trap lines demonstrated a defect in olfactory-driven behaviour. Further characterisation of these genes and their roles in olfactory system function and development will increase our understanding of how the insect olfactory system has evolved to perform the same essential function to that of mammals, but using very different molecular genetic mechanisms.