The remarkable responsiveness of male moths to female released pheromones is based on the extremely sensitive and selective reaction of highly specialized sensory cells in the male antennae. These cells are supposed to be equipped with male-specific receptors for pheromonal compounds, however, the nature of these receptors is still elusive. By using a combination of genomic sequence analysis and cDNA-library screening, we have cloned various cDNAs of the tobacco budworm Heliothis virescens encoding candidate olfactory receptors. A comparison of all identified receptor types not only highlighted their overall high degree of sequence diversity but also led to the identification of a small group of receptors sharing >40% identity. In RT-PCR analysis it was found that distinct members of this group were expressed exclusively in the antennae of male moths. In situ hybridization experiments revealed that the male-specific expression of these receptor types was confined to antennal cells located beneath sensillar hair structures (sensilla triochoidea), which have been shown to contain pheromone-sensitive neurons. Moreover, two-color double in situhybridization approaches uncovered that cells expressing one of these receptor types were surrounded by cells expressing pheromone-binding proteins, as expected for a pheromone-sensitive sensillum. These findings suggest that receptors like Heliothis receptor 14 -16 (HR14 -HR16) may render antennal cells responsive to pheromones.T he olfactory system of insects, most notably of moths, is renowned for its remarkable sensitivity and selectivity; it has been an invaluable model system for studying fundamental aspects of olfaction (1). Insects detect volatile chemostimulants by means of chemosensory neurons housing in multiporous cuticular hairs, comprising olfactory sensilla (2). These specialized cells generate electrical signals upon interaction with appropriate chemical compounds. Experimental evidence indicating that the underlying chemoelectrical transduction process is mediated by means of odor-activated G protein-secondmessenger cascades, a mechanism used by most chemosensory cells (3), supports the notion that receptors for odorous compounds in insects should be members of the G protein-coupled receptor superfamily; however, it was only with the aid of sequenced genomes that genes encoding candidate odorant receptors from insects were identified recently in fly (4-8), mosquito (9-11), and moth (12) models. Despite the progress in identification and characterization of insect olfactory receptors, receptors for insect pheromones are still elusive. This status is more noteworthy given that the pioneering work in identifying signaling molecules for communication between individuals was performed on insects; in fact, the first known pheromone (bombykol) was isolated from the silkmoth Bombyx mori (13,14). Pheromones, originally defined as chemical compounds that are produced and secreted by individuals and received by other members of the same species in which they release a de...

Candidate olfactory receptors of the moth Heliothis virescens were found to be extremely diverse from receptors of the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae, but there is one exception. The moth receptor type HR2 shares a rather high degree of sequence identity with one olfactory receptor type both from Drosophila (Dor83b) and from Anopheles (AgamGPRor7); moreover, in contrast to all other receptors, this unique receptor type is expressed in numerous antennal neurons. Here we describe the identification of HR2 homologues in two further lepidopteran species, the moths Antheraea pernyi and Bombyx mori, which share 86-88% of their amino acids. In addition, based on RT-PCR experiments HR2 homologues were discovered in antennal cDNA of the honey bee (Apis mellifera; Hymenoptera), the blowfly (Calliphora erythrocephala; Diptera) and the mealworm (Tenebrio molitor; Coleoptera). Comparison of all HR2-related receptors revealed a high degree of sequence conservation across insect orders. In situ hybridization of antennal sections from the bee and the blowfly support the notion that HR2-related receptors are generally expressed in a very large number of antennal cells. This, together with the high degree of conservation suggests that this unique receptor subtype may fulfill a special function in chemosensory neurons of insects.

The antennae of moths have been an invaluable model for studying the principles of odour perception. In spite of the enormous progress in understanding olfaction on the molecular level, for the moth one of the key elements in olfactory signalling, the odourant receptors, are still elusive. We have assessed a genome database of a heliothine moth (Heliothis virescens, Noctuidae) and employed exon-specific probes to screen an antennal cDNA library of this species. Analysis of isolated cDNA-clones led to the discovery of a divergent gene family encoding putative seven-transmembrane domain proteins. The notion that they may encode candidate olfactory receptors of the moth, was supported by a tissue-specific expression; several of the subtypes were exclusively expressed in antennae. By means of double-labelling in situ hybridization studies it was demonstrated that the receptors are indeed expressed in antennal sensory neurons; moreover, each receptor subtype appears to be expressed in a distinct population of sensory cells. The results strongly suggest that the newly discovered gene family indeed encodes olfactory receptors of moth.