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...

Males of the moth species Heliothis virescens are able to detect the female-released pheromone with remarkable sensitivity and specificity, distinguishing between highly related pheromonal compounds. In the past, electrophysiological studies succeeded in assigning sensory hairs to identified compounds revealing three functional types of long sensilla trichodea housing neurons specifically responding to distinct semiochemicals. The specific responsiveness implies that the sensory neurons express different receptor types tuned to pheromone components. In this study we demonstrate that heterologously expressed candidate pheromone receptors from Heliothis responded to several pheromonal compounds, including the major sex-pheromone component Z-11-hexadecenal indicating a limited specificity of each receptor type. Nonetheless, based on functional analysis and in situ hybridization studies the analysed receptor types could tentatively be assigned to types of long sensilla trichodea, containing the pheromone-binding proteins (PBPs) HvirPBP1 and HvirPBP2 in the sensillum lymph. Substituting organic solvent with PBPs to solubilize the hydrophobic pheromone compounds in functional assays revealed an increase in sensitivity and especially specificity. It was found that in the presence of HvirPBP2, cells expressing the receptor type HR13 specifically responded to the main component of the sex pheromone blend only. The data provide further evidence that a combination of a distinct receptor type and binding protein underlie the specific response observed in the detection of a pheromone component in vivo.

In moths the detection of female-released sex pheromones involves hairlike structures on the male antenna. These long sensilla trichodea usually contain 2-3 chemosensory neurons accompanied by several supporting cells. Previous studies have shown that the pheromone-specific neurons are characterized by a "sensory neuron membrane protein" (SNMP) which is homologous to the CD36 family and localized in the dendrite membrane. By employing the SNMP-2 sequence from Manduca sexta we have isolated cDNAs that encode SNMP-2 proteins from Heliothis virescens (HvirSNMP-2) and Antheraea polyphemus (ApolSNMP-2). To elucidate the topographic and cell type-specific expression of these SNMP subtypes, 2-color in situ hybridization experiments were performed with tissue sections through the male antennae. For H. virescens, a specific probe for the pheromone receptor HR13 was used to identify pheromone-responsive neurons. It was found that HvirSNMP-1 and HR13 were coexpressed in the same cells; in contrast, HvirSNMP-2 was not expressed in HR13 cells but rather in cells that surrounded the HR13 neurons, apparently the supporting cells. A corresponding expression pattern was also found for ApolSNMP-1 and ApolSNMP-2 on the antenna of male A. polyphemus. Our results indicate that SNMP-1s and SNMP-2s are differentially expressed in cells of pheromone-sensitive sensilla and suggest distinct functions for the 2 SNMP subtypes in the olfactory system.

Communication via specific chemical signals is vitally important for the reproductive behaviour of many species. The first identified sex-attractant pheromone was bombykol from the silkmoth Bombyx mori. This female-released signalling compound is perceived by the male moth with extreme sensitivity and specificity. Antennal sensory cells supposedly respond to individual bombykol molecules and can efficiently distinguish bombykol from highly related structural analogues like bombykal, a second female-released pheromone component. In the four decades since the discovery of bombykol, the Bombyx mori system has continued to serve as an invaluable model system for unraveling the intricacies of chemical communication. The molecular basis for this extraordinary specific recognition of an extraneous compound is still elusive but probably based on specific receptors of the pheromone-responsive cells. In this study, molecular and bioinformatic approaches were employed to search for candidate pheromone receptors of Bombyx mori. A few receptor types were identified that are related to Heliothis candidate pheromone receptors. They were found to be almost exclusively expressed in male antennae, and double in situ hybridization experiments disclosed a characteristic topographic expression pattern that was reminiscent of pheromone-responsive cells. Furthermore, the receptor-expressing cells were closely associated with cells expressing the pheromone-binding protein. Together, the data support the view that the identified receptor types of Bombyx mori are candidate receptors for pheromone components.