Gustatory organs of Drosophila melanogaster: fine structure and expression of the putative odorant-binding protein PBPRP2

In Drosophila, as in most insects, gustation is mediated by sensory hairs located on the external and internal parts of the proboscis and on the legs and wings. We describe in detail the organization and ultrastructure of the gustatory sensilla on the labellum and legs and the distribution of PBPRP2, a putative odorant-binding protein, in the gustatory organs of Drosophila. The labellum carries two kinds of sensilla: taste bristles and taste pegs. The former have the typical morphology of gustatory sensilla and can be further subdivided into three morphological subtypes, each with a stereotyped distribution and innervation. Taste pegs have a unique morphology and are innervated by two receptor cells: one mechanoreceptor and the other a putative chemoreceptor cell. PBPRP2 is abundantly expressed in all adult gustatory organs on labellum, legs, and wings and in the internal taste organs on the proboscis. In contrast to olfactory organs, where PBPRP2 is expressed in the epidermis, this protein is absent from the epidermis of labial palps and legs. In the taste bristles of the labellum and legs, PBPRP2 is localized in the crescent-shaped lumen of the sensilla, and not in the lumen where the dendrites of the gustatory neurons are found, making a function in stimulus transport unlikely in these sensilla. In contrast, PBPRP2 in peg sensilla is expressed in the inner sensillum-lymph cavity and is in contact with the dendrites. Thus, PBPRP2 could be involved as a carrier for hydrophobic ligands, e.g., bitter tastants, in these sensilla.     

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.     

A large family of divergent Drosophila odorant-binding proteins expressed in gustatory and olfactory sensilla.

We identified a large family of putative odorant-binding protein (OBP) genes in the genome of Drosophila melanogaster. Some of these genes are present in large clusters in the genome. Most members are expressed in various taste organs, including gustatory sensilla in the labellum, the pharyngeal labral sense organ, dorsal and ventral cibarial organs, as well as taste bristles located on the wings and tarsi. Some of the gustatory OBPs are expressed exclusively in taste organs, but most are expressed in both olfactory and gustatory sensilla. Multiple binding proteins can be coexpressed in the same gustatory sensillum. Cells in the tarsi that express OBPs are required for normal chemosensation mediated through the leg, as ablation of these cells dramatically reduces the sensitivity of the proboscis extension reflex to sucrose. Finally, we show that OBP genes expressed in the pharyngeal taste sensilla are still expressed in the poxneuro genetic background while OBPs expressed in the labellum are not. These findings support a broad role for members of the OBP family in gustation and olfaction and suggest that poxneuro is required for cell fate determination of labellar but not pharyngeal taste organs.     

Internalization of odorant-binding proteins into the mouse olfactory epithelium

The detection of odorants in vertebrates is mediated by chemosensory neurons that reside in the olfactory epithelium of the nose. In land-living species, the hydrophobic odorous compounds inhaled by the airstream are dissolved in the nasal mucus by means of specialized globular proteins, the odorant-binding proteins (OBPs). To assure the responsiveness to odors of each inhalation, a rapid removal of odorants from the microenvironment of the receptor is essential. In order to follow the fate of OBP/odorant complexes, a recombinant OBP was fluorescently labeled, loaded with odorous compounds, and applied to the nose of a mouse. Very quickly, labeled OBP appeared inside the sustentacular cells of the epithelium. This uptake occurred only when the OBP was loaded with appropriate odorant compounds. A search for candidate transporters that could mediate such an uptake process led to the identification of the low density lipoprotein receptor Lrp2/Megalin. In the olfactory epithelium, megalin was found to be specifically expressed in sustentacular cells and the Megalin protein was located in their microvilli. In vitro studies using a cell line that expresses megalin revealed a rapid internalization of OBP/odorant complexes into lysosomes. The uptake was blocked by a Megalin inhibitor, as was the internalization of OBPs into the sustentacular cells of the olfactory epithelium. The results suggest that a Megalin-mediated internalization of OBP/odorant complexes into the sustentacular cells may represent an important mechanism for a rapid and local clearance of odorants.     

Preferential expression of biotransformation enzymes in the olfactory organs of Drosophila melanogaster, the antennae

Biotransformation enzymes have been found in the olfactory epithelium of vertebrates. We now show that in Drosophila melanogaster, a UDP-glycosyltransferase (UGT), as well as a short chain dehydrogenase/reductase and a cytochrome P450 are expressed specifically or preferentially in the olfactory organs, the antennae. The evolutionarily conserved expression of biotransformation enzymes in olfactory organs suggests that they play an important role in olfaction. In addition, we describe five Drosophila UGTs belonging to two families. All five UGTs contain a putative transmembrane domain at their C terminus as is the case for vertebrate UGTs where it is required for enzymatic activity. The primary sequence of the C terminus, including part of the transmembrane domain, differs between the two families but is highly conserved not only within each Drosophila family, but also between the members of one of the Drosophila families and vertebrate UGTs. The partial overlap of the conserved primary sequence with the transmembrane domain suggests that this part of the protein is involved in specific interactions occurring at the membrane surface. The presence of different C termini in the two Drosophila families suggests that they interact with different targets, one of which is conserved between Drosophila and vertebrates.     

Two thermosensors in Drosophila have different behavioral functions

Insects inhabit extreme temperature environments and have evolved mechanisms to survive there. Small insects are especially susceptible to rapid changes in body temperature. Therefore, the rapid detection of environment and body temperature is important for their survival. Little, however, is known about the thermosensors that detect those temperatures. Using rapid thermosensitivity assays with temperature step gradients and a spatial learning paradigm (the heat-box) in which elevated temperature serves as the negative reinforcer, two thermosensors were identified and their behavioral functions assessed. A low-temperature thermosensor is located on the antenna, detects relatively low temperatures, and can detect spatial temperature gradients directly. Thus, the antennae can be used by Drosophila to quickly orient with respect to temperature cues. A high-temperature thermosensor of unknown location appears to have a roughly similar sensitivity to temperature differences as the low-temperature thermosensor (< or = 3 degrees C) and is both necessary and sufficient for memory formation in the heat-box spatial learning paradigm. Therefore, the high-temperature thermosensor is important for remembering spatial positions in which dangerously high temperatures were encountered.     

The invertebrate odorant-binding protein LUSH is required for normal olfactory behavior in Drosophila

The invertebrate odorant-binding proteins consist of a large family of low-molecular-weight, highly divergent proteins expressed exclusively in the chemosensory sensilla of insects. Each member of this family studied to date is secreted into the sensillum lymph of a small subset of sensilla by non-neuronal support cells. These expression patterns suggests an odor-specific function for these proteins as opposed to a general role in sensillum biology. Consistent with this notion, mutants defective for LUSH, a Drosophila member of this family, have odor-specific defects in olfactory behavior. The Drosophila genome contains at least 32 members of this gene family, rivaling the number of odorant receptors in this species. The relationship between these two protein families and how they act to determine odor specificity of olfactory neurons will be the topic of future studies.     

Microtubule-associated proteins present in different developmental stages of Drosophila melanogaster

Microtubule-associated proteins (MAPs) have been isolated from different development stages of Drosophila melanogaster and characterized by their association to tubulin, but not to tubulin lacking its 4-kD carboxy terminal region (S-tubulin), and by their ability to promote tubulin polymerization. Following these criteria some peptides of Mr 255, 205, and 180 kD were identified as MAPs. By means of immunological analogy we have identified a peptide related to mammalian brain MAP known as tau factor.     

An overview of odorant-binding protein functions in insect peripheral olfactory reception

Insect olfactory perception involves many aspects of insect life, and can directly or indirectly evoke either individual or group behaviors. Insect olfactory receptors and odorant-binding proteins (OBPs) are considered to be crucial to insect-specific and -sensitive olfaction. Although the mechanisms of interaction between OBPs or OBP/ligand complex with olfactory receptors are still not well understood, it has been shown that many OBPs contribute to insect olfactory perception at various levels. Some of these are numerous and divergent members in OBP family; expression in the olfactory organ at high concentration; a variety of combinational patterns between different OBPs and ligands, but exclusive affinity for one OBP to specific binding ligands; complicated interactions between OBP/ligand complex and transmembrane proteins (olfactory receptors or sensory neuron membrane proteins). First, we review OBPs' ligand-binding property based on OBP structural research and ligand-binding test; then, we review current progress around the points cited above to show the role of such proteins in insect olfactory signal transmission; finally, we discuss applications based on insect OBP research.     

A putative pheromone receptor gene is expressed in two distinct olfactory organs in goats

Mammals possess two anatomically and functionally distinct olfactory systems. The olfactory epithelium (OE) detects volatile odorants, while the vomeronasal organ (VNO) detects pheromones that elicit innate reproductive and social behavior within a species. In rodent VNO, three multigene families that encode the putative pheromone receptors, V1Rs, V2Rs and V3Rs, have been expressed. We have identified the V1R homologue genes from goat genomic DNA (gV1R genes). Deduced amino acid sequences of gV1R genes show 40-50% and 20-25% identity to those of rat and mouse V1R and V3R genes, respectively, suggesting that the newly isolated goat receptor genes are members of the V1R gene family. One gene (gV1R1 gene) has an open reading frame that encodes a polypeptide of 309 amino acids. It is expressed not only in VNO but also in OE. In situ hybridization analysis revealed that gV1R1 -expressing cells were localized in neuropithelial layers of VNO and OE. These results suggest that the goat may detect pheromone molecules through two distinct olfactory organs.     

Nested hierarchal organization of conservation for microRNAs and their putative targets to Drosophila melanogaster

This study examined microRNA network properties traced through taxonomic hierarchy considering both the acquisition of potential network targets and regulators. Primary literature review and database analyses were conducted to establish modules of conserved microRNAs across metazoan taxonomy. A hierarchical schema for the conservation of microRNAs and their putative targets to Drosophila melanogaster was engineered through comprehensive meta-analysis, and conservation history of 90.39% of the total Drosophila dataset could be resolved through this hierarchical sampling regime; tracing from taxonomic order down to empire. The findings presented in this study represent a documentation of Drosophila microRNA regulatory network behavior thorough taxonomic hierarchy. MicroRNA regulatory network properties were found to transect taxonomic hierarchy. Newly acquired microRNAs from novel families reinforce the pre-existing regulatory network, while expanding the target list to include a small number of novel genes. Lineage specific microRNAs were found to exhibit far fewer conserved targets than do the more broadly conserved microRNAs; even when considering only more recently emerged targets. There was a dramatic expansion in network complexity with the expansion of the microRNA repertoire, and this corresponds to the expansion in biological complexity.     

Expression of odorant-binding proteins and chemosensory proteins in some Hymenoptera

The expression of chemosensory proteins (CSPs) and odorant-binding proteins (OBPs) in individuals of different castes and ages have been monitored in three species of social hymenopterans, Polistes dominulus (Hymenoptera, Vespidae), Vespa crabro (Hymenoptera, Vespidae) and Apis mellifera (Hymenoptera, Apidae), using PCR with specific primers and polyclonal antibodies. In the paper wasp P. dominulus, OBP is equally expressed in antennae, wings and legs of all castes and ages, while CSP is often specifically present in antennae and in some cases also in legs. In the vespine species V. crabro CSP is antennal specific, while OBP is also expressed in legs and wings. The three CSPs and the five OBPs of A. mellifera show a complex pattern of expression, where both classes of proteins include members specifically expressed in antennae and others present in other parts of the body. These data indicate that at least in some hymenopteran species CSPs are specifically expressed in antennae and could perform roles in chemosensory perception so far assigned only to OBPs.     

Experimental evolution of olfactory memory in Drosophila melanogaster

In order to address the nature of genetic variation in learning performance, we investigated the response to classical olfactory conditioning in "high-learning" Drosophila melanogaster lines previously subject to selection for the ability to learn an association between the flavor of an oviposition medium and bitter taste. In a T-maze choice test, the seven high-learning lines were better at avoiding an odor previously associated with aversive mechanical shock than were five unselected "low-learning" lines originating from the same natural population. Thus, the evolved improvement in learning ability of high-learning lines generalized to another aversion learning task involving a different aversive stimulus (shock instead of bitter taste) and a different behavioral context than that used to impose selection. In this olfactory shock task, the high-learning lines showed improvements in the learning rate as well as in two forms of consolidated memory: anesthesia-resistant memory and long-term memory. Thus, genetic variation underlying the experimental evolution of learning performance in the high-learning lines affected several phases of memory formation in the course of olfactory aversive learning. However, the two forms of consolidated memory were negatively correlated among replicate high-learning lines, which is consistent with a recent hypothesis that these two forms of consolidated memory are antagonistic.     

Electrophoretic patterns of chorion proteins and their relation to phylogeny of eleven Drosophila species of the melanogaster group

The electrophoretic patterns of chorion proteins coded by four chorion genes in eleven Drosophila species of the melanogaster group have been studied. We found that, in spite of the specific characteristics of this unique set of genes, the electrophoretic patterns are, in general, in accordance to the proposed phylogenies.     

Expression patterns of two new members of the Semaphorin family in Drosophila suggest early functions during embryogenesis

We report the sequence and expression analysis of two new Drosophila members of the Semaphorin family. Both proteins show the presence of Semaphorin domains and transmembrane domains. Both genes are expressed maternally and in embryos, and reveal distinct expression patterns much earlier than the onset of neurogenesis. We also present an overview of the domain structure of all so far known semaphorins in Drosophila. Furthermore, we compared all Drosophila and C. elegans Semaphorins and discuss them in the light of their evolution.     

Evolution of the olfactory code in the Drosophila melanogaster subgroup

The Drosophila melanogaster subgroup has been the focus of numerous studies about evolution. We address the question of how the olfactory code has evolved among the nine sister species. By using in vivo electrophysiological measurements, so called single-cell recordings, we have established the ligand affinity of a defined subset of olfactory receptor neurons (ORNs) across all nine species. We show that the olfactory code as relayed by the investigated subset of ORNs is conserved to a striking degree. Distinct shifts in the code have occurred only within the simulans clade. However, these shifts are restricted to an altered tuning profile of the same single ORN type in all three of the simulans siblings and a more drastic change unique to D. sechellia, involving a complete loss of one sensillum type in favour of another. The alterations observed in D. sechellia may represent a novel host-specific adaptation to its sole host, morinda fruit (Morinda citrifolia). The overall high degree of similarity of the code within the subgroup is intriguing when considering the great variety in distributions as well as in habitat and host choice of the siblings, factors that could greatly affect the olfactory system.     

Revisiting the odorant-binding protein LUSH of Drosophila melanogaster: evidence for odour recognition and discrimination

LUSH is a soluble odorant-binding protein of the fruit fly Drosophila melanogaster. Mutants not expressing this protein have been reported to lack the avoidance behaviour, exhibited by wild type flies, to high concentrations of ethanol. Very recently, the three-dimensional structure of LUSH complexed with short-chain alcohols has been resolved supporting a role for this protein in binding and detecting small alcohols. Here we report that LUSH does not bind ethanol and that wild type flies are in fact attracted by high concentrations of ethanol. We also report that LUSH binds some phthalates and that flies are repelled by such compounds. Finally, our fluorescence data, interpreted in the light of the three-dimensional structure of LUSH, indicate that the protein undergoes a major conformational change, similar to that reported for the pheromone-binding protein of Bombyx mori, but triggered, in our case, by ligand.     

Drosophila melanogaster p24 trafficking proteins have vital roles in development and reproduction

p24 proteins comprise a family of type-I transmembrane proteins of ~24kD that are present in yeast and plants as well as metazoans ranging from Drosophila to humans. These proteins are most commonly localized to the endoplasmic reticulum (ER)-Golgi interface and are incorporated in anterograde and retrograde transport vesicles. Little is known about how disruption of p24 signaling affects individual tissue function or whole animals. Drosophila melanogaster express nine p24 genes, grouped into four subfamilies. Based upon our mRNA and protein expression data, Drosophila p24 family members are expressed in a variety of tissues. To identify functions for particular Drosophila p24 proteins, we used RNA interference (RNAi) to reduce p24 expression. Ubiquitous reduction of most p24 genes resulted in complete or partial lethality during development. We found that reducing p24 levels in adults caused defects in female fecundity (egg laying) and also reduced male fertility. We attributed reduced female fecundity to decreased neural p24 expression. These results provide the first genetic analysis of all p24 family members in a multicellular animal and indicate vital roles for Drosophila p24s in development and reproduction, implicating neural expression of p24s in the regulation of female behavior.