Olfaction in insects is remarkably sensitive. In social insects, like the honeybee, pheromonal communication is the key to controlling colony integration and survival. OBPs (odourant binding proteins) are believed to be the first biochemical step in the recognition of odourants such as pheromones. They are proposed to facilitate transportation of lipophilic odourant molecules through the hydrophilic sensillar lymph to membrane bound receptors on the olfactory neurones. The heterogeneity of OBPs found in insects suggests that they play a role in odour discrimination.
In this study, I have characterised two genes identified from the antennal cDNA library, one of which encodes an odourant binding protein while the other encodes a putative chemosensory protein. The ASP1 (putative pheromone binding protein) has the characteristic hallmark of insect OBPs, for example, the signal sequence, acidic isoelectric point and the six conserved cysteine residues. ASP3c belongs to the chemosensory protein family which has similar characteristics to the OBP family but with only four conserved cysteines. Southern hybridisation indicates only one copy of each gene exists in the genome.
I have also identified seventeen candidate OBP genes in the Drosophila genome. The phylogenetic relationships between them and other OBPs indicates that the insect OBP family is large and very diverse. It is likely that OBPs are present throughout all species of insect, though they are notably absent from C. elegans which is a phylogenetically related member of the Ecdysozoa. Only one novel member of the chemosensory protein family was identified in the Drosophila genome indicating that this protein family is not as large as the OBP family.
Northern hybridization was used to identify correlation between gene expression and caste and age of honeybees. In these studies, drones were found to have 2-3 fold higher ASP1 transcript levels than worker bees, which is consistent with its published role as a queen pheromone binding protein. Age related expression of the ASP1 gene does not correlate well with age-related behaviour of the honeybee. For example, mature drones which are highly sensitive to queen pheromone have a slightly lower ASP1 transcript level than immature drones. In addition, there is no change in ASP1 transcription level in worker bees when they are deprived of exposure to queen pheromone. Unlike ASP1, ASAP3c gene expression is strongly correlated with age. Newly emerged drones and worker bees both have maximum levels of ASP3c transcript. This suggests an important role of ASP3c in chemosensory process that is uniquely perceived by young bees within the environment of the hive.
ASP1 antibodies against synthetic peptides and native protein purified by polyacrylamide gel electrophoresis were produced. The antibody against native protein had a higher activity than the antibody against synthetic peptide. It was also found to be very specific as no other proteins from other olfactory organs such as maxillary palps or legs showed any immuno-crossreactivity to the ASP1 antibody. The limited reaction of ASP1 antibody to proteins from the scape and pedicel of the antennae probably reflects the small number of sensilla trichodea (s. trichodea) present in these fi-agments. The queen pheromone producing glands, such as the mandibular gland, tergites and tarsi of queens also contain no proteins that cross-react with the ASP1 antibody. Immunohistochemical localisation of ASP1 to both s. placodea and some fraction of s. trichodea indicates the function of ASP1 in olfaction. S. coeloconica , s. ampulacea and some fraction of s. trichodea have no labeling.