Genes encoding candidate pheromone receptors in a moth (Heliothis virescens)

Abstract

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 situ-hybridization 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.

Fig. 1.

Neighbor-joining tree showing the sequence relatedness between putative chemosensory receptors in H. virescens. The distance tree was calculated by using the mega program and is based on a clustal alignment of HR1–HR9 (12) and HR10–HR21 (this study). Branch lengths are proportional to the percentage of sequence difference. (Scale bar, 10% difference.) Boot-strap-support values (only >75% shown) are based on 1,000 replicates. The tree is rooted at the midpoint.

Fig. 2.

Tissue specificity of chemosensory receptor expression. RT-PCRs were performed with specific primer pairs and cDNAs prepared from different Heliothis tissues. Reaction products were visualized by ethidium bromide staining and UV illumination. Bands were of the expected size based on the primer design. HR14–HR16 expression was detected only in the antennae of males. The results for HR11 indicate differences in the expression levels between sexes. HR13 was predominantly found in male antennae, but RNA for this receptor was detected also in other tissues. For HR6, very high levels of RNA were detected in antennae; lower levels were found in several other tissues. Bands of similar intensities obtained with primers specific to the RL31 control indicate the integrity of the various cDNA preparations. Am, antenna male; Af, antenna female; H, head without appendixes; P, proboscis; L, leg; W, wing; T, thorax; Ab, abdomen; M, muscle. The position of marker bands (bp) is indicated on the left.

Fig. 3.

Expression of specific receptor types in the antenna of H. virescens. DIG-labeled antisense RNA probes for HR13 (A and B), HR14 (C), HR15 (D), and HR16 (E) were hybridized to longitudinal cryosections through the antennae of male moths and visualized by fluorescence detection using HNPP/Fast Red. A and C–E show sagittal planes; B displays a horizontal plane. Pictures represent optical sections selected from a stack of confocal images generated by a laser-scanning microscope. The fluorescence channel is overlaid with the transmitted-light channel. Arrows indicate labeled cells. (Scale bars, 50 μm.)

Fig. 4.

Expression of PBP in the antenna of a male H. virescens. In situ hybridization was performed on a longitudinal cryosection with biotin-labeled antisense RNA. Hybridization signals were visualized by a detection system, indicating PBP-positive cells by green fluorescence. The image represents an optical section selected from a stack of confocal images that were generated by a laser-scanning microscope. Two segments of the filamentous antenna are shown. Hybridization signals can be assigned to cells lying under rows of long sensilla trichoidea; no labeling was detected on the scaled side. (Scale bar, 50 μm.)

Fig. 5.

Gene expression of HR15 and PBP in the antenna. Two-color in situ hybridization was performed on a section through the male antenna by using a combination of DIG-labeled HR15 and biotin-labeled PBP antisense RNAs. Hybridization signals were visualized by detection systems, indicating cells bearing HR15 transcripts by red fluorescence and PBP-positive cells in green. The side of two antennal segments carrying the sensilla hairs is shown. Images represent selected confocal images from a stack of confocal images that were taken with a laser-scanning microscope. Cells expressing HR15 were found to be accompanied by cells expressing PBP. (Scale bar, 50 μm.)

Fig. 6.

Colocalization of cells expressing the HR16 receptor type and PBP. Two-color in situ hybridization performed with DIG-labeled HR16 and biotin-labeled PBP antisense RNAs on a section through the male antenna. Hybridization signals were visualized by detection systems indicating cells bearing HR16 transcripts by red fluorescence and PBP-positive cells in green. All images show different optical planes of the section in an area just below the surface carrying the sensilla hairs with cells expressing HR16 or PBP. Images represent selected confocal images from a stack taken with a laser-scanning microscope. By following the sequence of images, it is obvious that cells expressing PBP surround a HR16-expressing cell. (Scale bar, 10 μm.)