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Four recombinant odorant-binding proteins of Bombyx mori, pheromone-binding protein (PBP), general odorant-binding protein 1 (GOBP1), general odorant-binding protein 2 (GOBP2) and antennal binding protein X (ABPX), were expressed in E. coli and used to raise polyclonal antisera. Immunoblots of antennal homogenates showed that these antisera were specific. In Western blot analysis and immunocytochemical labelling experiments, the sera against recombinant PBP and GOBP2 of B. mori gave identical results as sera against native PBP and GOBP2 of Antheraea polyphemus, respectively, thus confirming earlier results obtained with the latter. Labelling consecutive cross sections of various sensillum types with all four antisera revealed different labelling patterns in male and female sensilla (s.) trichodea and s. basiconica. Long s. trichodea in males and females represented uniform labelling types, whereas for short s. trichodea, s. intermedia, and s. basiconica a great variety of labelling patterns was observed, some being more common than others. Long s. trichodea, which in males are uniformly tuned to the pheromone components bombykol and bombykal, all strongly expressed PBP; labelling with antisera against the other three odorant-binding proteins hardly was above background, only in some hairs GOBP1 was expressed somewhat more strongly. Long s. trichodea of females, which respond specifically to linalool and benzoic acid, showed a different labelling pattern. Here, we observed strong labelling with antibodies against GOBP2 and medium labelling with anti-GOBP1, sometimes with anti-ABPX. S. basiconica in both sexes most commonly co-expressed GOBP1 and GOBP2, but other patterns were occasionally found, with some of them showing PBP expression, also in females. The great variety of labelling types in short s. trichodea, s. intermedia, and s. basiconica suggests a similar variety of functional subtypes as observed in plant odour-sensitive sensilla of other moth species.

Binding properties of six heterologously expressed pheromone-binding proteins (PBPs) identified in the silkmoths Antheraea polyphemus and Antheraea pernyi were studied using tritium-labelled pheromone components, ( E, Z)-6,11-hexadecadienyl acetate ((3)H-Ac1) and ( E, Z)-6,11-hexadecadienal ((3)H-Ald), common to both species. In addition, a known ligand of PBP and inhibitor of pheromone receptor cells, the tritium-labelled esterase inhibitor decyl-thio-1,1,1-trifluoropropanone ((3)H-DTFP), was tested. The binding of ligands was measured after native gel electrophoresis and cutting gel slices. In both species, PBP1 and PBP3 showed binding of (3)H-Ac1. In competition experiments with (3)H-Ac1 and the third unlabelled pheromone component, ( E, Z)-4,9-tetradecadienyl acetate (Ac2), the PBP1 showed preferential binding of Ac1, whereas PBP3 preferentially bound Ac2. The PBP2 of both species bound (3)H-Ald only. All of the six PBPs strongly bound (3)H-DTFP. Among unlabelled pheromone derivatives, alcohols were revealed to be the best competitors for (3)H-Ac1 and (3)H-Ald bound to PBPs. No pH influence was found for (3)H-Ac1 binding to, or its release from, the PBP3 of A. polyphemus and A. pernyi between pH 4.0 and pH 7.5. The data indicate binding preference of each of the three PBP-subtypes (1-3) for a specific pheromone component and support the idea that PBPs contribute to odour discrimination, although to a smaller extent than receptor activation.

Females of the sibling silkmoth species Antheraea polyphemus and A. pernyi use the same three sex pheromone components in different ratios to attract conspecific males. Accordingly, the sensory hairs on the antennae of males contain three receptor cells sensitive to each of the pheromone components. In agreement with the number of pheromones used, three different pheromone‐binding proteins (PBPs) could be identified in pheromone‐sensitive hairs of both species by combining biochemical and molecular cloning techniques. MALDI‐TOF MS of sensillum lymph droplets from pheromone‐sensitive sensilla trichodea of male A. polyphemus revealed the presence of three major peaks with m/z of 15702, 15752 and 15780 and two minor peaks of m/z 15963 and 15983. In Western blots with four antisera raised against different silkmoth odorant‐binding proteins, immunoreactivity was found only with an anti‐(Apol PBP) serum. Free‐flow IEF, ion‐exchange chromatography and Western blot analyses revealed at least three anti‐(Apol PBP) immunoreactive proteins with pI values between 4.4 and 4.7. N‐Terminal sequencing of these three proteins revealed two proteins (Apol PBP1a and Apol PBP1b) identical in the first 49 amino acids to the already known PBP (Apol PBP1) [Raming, K., Krieger, J. & Breer, H. (1989) FEBS Lett.256, 2215–2218] and a new PBP having only 57% identity with this amino‐acid region. Screening of antennal cDNA libraries with an oligonucleotide probe corresponding to the N‐terminal end of the new A. polyphemus PBP, led to the discovery of full length clones encoding this protein in A. polyphemus (Apol PBP3) and in A. pernyi (Aper PBP3). By screening the antennal cDNA library of A. polyphemus with a digoxigenin‐labelled A. pernyi PBP2 cDNA [Krieger, J., Raming, K. & Breer, H. (1991) Biochim. Biophys. Acta1088, 277–284] a homologous PBP (Apol PBP2) was cloned. Binding studies with the two main pheromone components of A. polyphemus and A. pernyi, the (E,Z)‐6,11‐hexadecadienyl acetate (AC1) and the (E,Z)‐6,11‐hexadecadienal (ALD), revealed that in A. polyphemus both Apol PBP1a and the new Apol PBP3 bound the 3H‐labelled acetate, whereas no binding of the 3H‐labelled aldehyde was found. In A. pernyi two PBPs from sensory hair homogenates showed binding affinity for the AC1 (Aper PBP1) and the ALD (Aper PBP2), respectively.

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