Molecular basis of female-specific odorant responses in Bombyx mori

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Abstract

Males and females of many moth species exhibit important differences in sexual behaviours. Much research in this field has focused on the male-specific behaviour, electrophysiology and molecular biology of sex pheromone reception. Female-specific behaviours have been less well studied although, like male-specific behaviours, they could provide opportunities for intervention and management of lepidopteran pests. Previously, we identified genes encoding putative odorant receptors (ORs) from the genome of the silkworm, Bombyx mori, some of which have higher levels of steady-state transcript in the antennae of adult females compared with males. We have identified the full-length cDNA sequences of some of these ORs and described a novel OR that is part of a female-biased clade. Using expression in Sf9 cells and a calcium-imaging assay, we tested a range of compounds for their ability to activate the most highly female-biased ORs, BmOR19, BmOR30, BmOR45 and BmOR47. BmOR19 responds to linalool, while BmOR45 and BmOR47 respond to benzoic acid > 2-phenylethanol > benzaldehyde. No activating ligands were found for BmOR30. RNA in situ hybridisation experiments reveal that BmOR19 is expressed in female olfactory sensory neurons that are co-located in the same sensilla as a second ORN expressing BmOR45 and/or BmOR47. Taken together the activity and expression of these receptors is likely explanatory of the observed electrophysiology of long sensilla trichoidea of female B. mori, previously shown to each contain one terpene (BmOR19) and one benzoic acid (BmOR45, BmOR47) sensory neuron. Plant volatiles such as linalool, benzoic acid, 2-phenylethanol and benzaldehyde are oviposition cues for females of some moths. These compounds have also been found in male-produced pheromone blends extracted from the hair pencils of many noctuid species. Hair pencil structures have not previously been reported for B. mori, but we have found hair pencil-like structures in adult male B. mori that are absent in female moths. It is proposed that BmOR19, BmOR45 and BmOR47 account for some of the female-specific odorant responses in B. mori, such as oviposition and/or detection of an as yet unidentified male-produced sex pheromone.

Introduction

The silkworm, Bombyx mori, is an important model system for studying lepidopteran and insect olfaction, with most research to date focusing on the detection by males of the female-produced sex pheromone components, bombykol and bombykal (Schneider, 1992). The antennae of B. mori are sexually dimorphic. Male antennae have slightly longer branches and many more olfactory hairs than female antennae (Heinbockel and Kaissling, 1996). Long sensilla trichoidea are the most abundant sensillum type. Each of the long trichoid sensilla in males contain two sensory neurons, one specifically responsive to bombykol and the other to bombykal (Kaissling et al., 1978). These neurons project to enlarged glomeruli found in the macroglomerular complex in the male (but not female) antennal lobe (Hildebrand and Shepherd, 1997). Functional assays in a heterologous system have shown the molecular basis of the odorant specificity is inherent to the odorant receptors, BmOR1 and BmOR3, that respond to bombykol and bombykal, respectively (Sakurai et al., 2004, Nakagawa et al., 2005).

The molecular basis of female-specific olfactory responses in B. mori is currently unknown. Long trichoid sensilla are also the most abundant sensillum type on female antennae, and also contain two neurons, however they don't respond to bombykol or bombykal (Boeckh et al., 1965). One neuron is highly sensitive to benzoic acid, while the other is sensitive to both 2,6-dimethyl-5-hepten-2-ol and linalool (Priesner, 1979, Heinbockel and Kaissling, 1996). These neurons project to large lateral glomeruli, which are enlarged in the female antennal lobe compared to the male (Koontz and Schneider, 1987). The behavioural significance of these specific odorant responses is not clear. Terpenoids (such as 2,6-dimethyl-5-hepten-2-ol and linalool) are common plant volatiles and it has been suggested that they may be used by female moths as indicators of host plant suitability and appropriate oviposition sites (Rostelien et al., 2005). The presence of many trichoid sensilla tuned to benzoic acid is even more puzzling. Research into odour stimulants occurring naturally in the habitat of B. mori showed that although the meconium of adult moths can activate the benzoic acid cell there is no behavioural response from females when exposed to meconium or benzoic acid (Heinbockel, 1990, Heinbockel and Kaissling, 1990).

The receptors responsible for these female-specific odorant responses have not been identified, however odorant receptors (ORs) that are over-expressed in female antennae compared to male antennae would be good candidates for this role. Recently, we identified 41 novel genes encoding putative ORs (Wanner et al., 2007), and a conserved lepidopteran receptor (Jordan et al., in press), bringing the total number of ORs known for B. mori to 49. Six of these ORs (BmOR12, BmOR19, BmOR30, BmOR45, BmOR46, BmOR47) have higher levels of steady-state transcript in the antennae of adult females compared with males. Two ORs are almost exclusively expressed in adult female antennae (BmOR19 and BmOR30; Wanner et al., 2007). Here we describe the characterisation of the ligand responsiveness of the four most highly female-biased ORs by heterologous expression in a lepidopteran cell line. We also demonstrate the in vivo cellular expression pattern of a subset of these receptors by RNA in situ hybridisation.

Section snippets

Materials

B. mori eggs from the domesticated Dazao strain were provided by Professor Q.-Y. Xia, South West University, China, and larvae were raised at 25 °C on Black mulberry leaves, Morus nigra, available locally in the summer months. All of the reagents and components used in assays were purchased from Sigma, unless stated otherwise. Compounds were purchased at the highest possible purity and included α-pinene, α-terpineol, p-cresol, 1,4-cineole, 2-phenylethanol, benzaldehyde, benzoic acid, butanal,

Female-biased odorant receptor gene isolation

BmOR19, BmOR30, BmOR45, and BmOR47 are female-specific or highly female-biased receptors in terms of expression at the RNA level within B. mori adult antennae. Therefore we obtained full-length cDNAs for these ORs for use in functional assays. cDNAs for BmOR19, BmOR30 and BmOR47 had some amino acid differences compared to the genomic sequences previously reported (GenBank accession numbers DQ991152, DQ991153, DQ991160) (Table 1). These differences most likely represent allelic polymorphisms.

Acknowledgements

We thank Liam Thammavongsa for insect rearing and Sylwek Chyb for reading the manuscript. This project was funded by CSIRO in Australia; The Foundation for Research, Science and Technology under contract numbers CO6X0701, CO6X0207, and C06X0301 and the Royal Society of New Zealand under the ISAT Linkages Fund (A06-42) in New Zealand; and USDA NRI grant #2007-35604-17756 in the USA.

References (28)

  • T. Heinbockel

    Variability of antennal receptor cell responses to benzoic acid and linalool in female Bombyx mori L.

  • T. Heinbockel et al.

    Sensitivity and inhibition of antennal benzoic acid receptor cells of female silkmoth Bombyx mori L.

    Verh. Dtsch. Zool. Ges.

    (1990)
  • J.G. Hildebrand et al.

    Mechanisms of olfactory discrimination: converging evidence for common principles across phyla

    Annu. Rev. Neurosci.

    (1997)
  • Jordan, M., Anderson, A.R., Begum, D., Carraher, C., Authier, A., Marshall, S.D.G., Kiely, A., Gatehouse, L.N.,...
  • Cited by (0)

    1

    These authors contributed equally to this manuscript.

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