6-11-hexadecadienal and 6-11-hexadecadienyl-acetate

While 11 species in the family Saturniidae are found in Japan, no sex pheromones of the native species had been investigated previously. We collected larvae of Rhodinia fugax in Nagano and Tottori Prefecture, and of Loepa sakaei in Okinawa Prefecture, and extracted sex pheromones of these two species from virgin female moths. In gas chromatography-electroantennogram detection (GC-EAD) analyses, male antennae of each species responded to one component in the respective pheromone extracts of conspecific females. Chemical analyses of the extracts by GC/mass spectrometry revealed that the EAD-active compounds of R. fugax and L. sakaei were a hexadecadienal and a tetradecadienyl acetate, respectively. The two species belong to the subfamily Saturniinae, and the mass spectra of both were similar to that of the 6,11-hexadecadienyl acetate identified from Antheraea polyphemus, classified in the same subfamily, suggesting the same 6,11-dienyl structure for the C16 aldehyde and a 4,9-dienyl structure for the C14 acetate. Based on this assumption, four geometrical isomers of each dienyl compound were stereoselectively synthesized via acetylene intermediates, compared to the natural products, and tested in the field. Male catches confirmed the pheromone structures of the two Japanese saturniid species as (6E,11Z)-6,11-hexadecadienal for R. fugax and (4E,9Z)-4,9-tetradecadienyl acetate for L. sakaei. The compounds have a characteristic 1,6-dienyl motif common to the pheromones of Saturniinae species.

Topics: Acetates; Alkadienes; Animals; Female; Gas Chromatography-Mass Spectrometry; Japan; Male; Molecular Structure; Moths; Pheromones; Sex Attractants

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.

Topics: Acetates; Aldehydes; Alkadienes; Animals; Binding, Competitive; Carrier Proteins; Chromatography, Gel; Chromatography, Ion Exchange; Cloning, Molecular; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Genetic Vectors; Hydrocarbons, Fluorinated; Hydrogen-Ion Concentration; Insect Proteins; Intercellular Signaling Peptides and Proteins; Ligands; Moths; Pheromones; Propane; Protein Binding; Recombinant Proteins; Tritium

One subtype of the pheromone binding proteins of the silkmoth Antheraea polyphemus (ApolPBP1) has been analysed exploiting the two endogenous tryptophan residues as fluorescent probe. The intrinsic fluorescence exhibited a rather narrow spectrum with a maximum at 336 nm. Site-directed mutagenesis experiments revealed that one of the tryptophan residues (Trp37) is located in a hydrophobic environment whereas Trp127 is more solvent exposed, as was predicted modeling the ApolPBP1 sequence on the proposed structure of the Bombyx mori pheromone binding protein. Monitoring the interaction of ApolPBP1 as well as its Trp mutants with the three species-specific pheromone compounds by recording the endogenous fluorescence emission revealed profound differences; whereas (E6,Z11)-hexadecadienal induced a dose-dependent quenching of the fluorescence, both (E6,Z11)-hexadecadienyl-1-acetate and (E4,Z9)-tetradecadienyl-1-acetate elicited an augmentation of the endogenous fluorescence. These data indicate that although ApolPBP1 can bind all three pheromones, there are substantial differences concerning their interaction with the protein, which may have important functional implications.

Topics: Acetates; Alkadienes; Animals; Bombyx; Carrier Proteins; Fluorescence; Insect Proteins; Intercellular Signaling Peptides and Proteins; Pheromones; Tryptophan

Female Attacus atlas respond electrophysiologically to both of the Antheraea polyphemus pheromone components (E,Z)-6,11-hexadecadienyl acetate and (E,Z)-6,11-hexadecadienal. Moreover, they possess a pheromone-binding protein (PBP) and general odorant-binding proteins (GOBPs), as well as a pheromone-degrading sensillar esterase and aldehyde oxidase enzymes. They show no electroantennogram responses to their own gland extract. In contrast, female A. polyphemus do not respond to their own or to A. atlas pheromone. Male A. atlas do not detect any of the A. polyphemus compounds but only the conspecific female gland extracts. Both male A. atlas and female A. polyphemus possess PBP and GOBP but lack the pheromone-degrading esterases of male Antheraea. The results indicate that the two species use quite distinct classes of chemicals as pheromones. In spite of this, the N-terminal amino acid sequences of the PBPs show homology of 68%.

Topics: Acetates; Alkadienes; Amino Acid Sequence; Animals; Carrier Proteins; Electrophysiology; Female; Insect Proteins; Intercellular Signaling Peptides and Proteins; Male; Molecular Sequence Data; Moths; Pheromones; Receptors, Odorant; Sequence Homology, Amino Acid; Species Specificity