Rosario Maida

The Pheromone Binding Protein of Bombyx mori: Purification, Characterization and Immunocytochemical Localization

Abstract A pheromone-binding protein (PBP) has been purified from the antennae of male Bombyx mori . It is a single polypeptide chain with a molecular weight of 16 kDa and an isoelectric point of 4.9. The PBP is present in the male and, to a minor extent, also in the female antennae, but not in other parts of the body. Strong crossreactivity has been observed with antibodies against the PBP of Antheraea polyphemus . Immunocytochemical localization indicates that the protein is concentrated in the sensillum lymph of sensilla trichodea.

Putative odorant-binding protein in antennae and legs of Carausius morosus (Insecta, Phasmatodea).

A 19 kDa protein has been purified by gel filtration and anion-exchange chromatography from the antennae of Carausius morosus. Its amino terminal amino acid sequence shows significant similarity (30% identity) with another putative odorant-binding protein, the so called OS-D protein isolated from the antennae of Drosophila melanogaster; only 20% of its amino acids are shared with some members of Lepidoptera pheromone-binding proteins. Polyclonal antibodies, raised against a synthetic amino terminal peptide cross-react with 19 kDa band in the legs extracts, but not with soluble proteins from other parts of the body. The amino terminal sequence of this protein, purified from the legs was identical with that of the antennal protein.

G-protein activation, identification and immunolocalization in pheromone-sensitive sensilla trichodea of moths.

Abstract.Electrophysiological in situ recordings from pheromone-sensitive sensilla trichodea of Bombyx mori males with a recording pipette which contained G-protein-activating fluoride, showed receptor cell activity similar to that evoked by pheromone stimulation. This suggests that G-proteins might be physiologically active in olfactory sensilla of insects in situ. Biochemical experiments using specific antibodies revealed the presence of G-protein, belonging to the Gq family, in antennal preparations. Similar G-protein was identified in sensory hair preparations of Antheraea pernyi which contained only cuticle, sensillum lymph and dendritic material. Moreover, the absence of this G-protein in pure sensillum lymph preparations indicates its association with the receptive dendrites. This particular association could be shown by immunolabelling studies at the ultrastructural level. Strong specific labelling of membranes of receptor-cell dendrites was found in all types of olfactory sensilla present on the antenna of the silkmoths. Additional specific labelling of apical membranes of auxiliary cells, epidermal cells and membranes forming the axon/glia interface demonstrated that this G-protein is not restricted to the sensory dendrites and that other signal-transduction pathways could be present at these membranes. In summary, the experiments imply a participation of G-protein of the Gq family in signal transduction of olfactory receptor cells in moths.

Identification of Plcβ and Pkc in pheromone receptor neurons of antheraea polyphemus

Two proteins of the IP3 transduction pathway were identified by Western blots in homogenates of isolated pheromonesensitive sensilla of the silkmoth Antheraea polyphemus. A 110 kDa protein was recognized by an antiserum raised against the Drosophila phospholipase Cβ (PLCβ p121) and a 80 kDa protein was labelled by an antiserum against a synthetic peptide of a conserved region of protein kinase C (PKC). Incubation of homogenized sensory hairs with the main sex pheromone component, (E,Z) 6–11 hexadecadienyl acetate, resulted in a 6-fold increase in the activity of PKC compared to controls without pheromone. In contrast, incubation with pheromone did not affect the activity of protein kinase A (PKA). Activation of PKC by the membrane permeable dioctanoylglycerol led to excitation of the pheromone-sensitive receptor neurons. These data support the current concept that pheromone perception of moths is mediated by the IP3 transduction pathway.

Esterase activity in the olfactory sensilla of the silkmoth Antheraea polyphemus

We studied in individual males of Antheraea polyphemus the activity of the sensillar esterase, a pheromone-degrading enzyme present in the sensillum lymph surrounding the olfactory receptor cells. In parallel, receptor potentials from single pheromone-sensitive sensilla trichodea were recorded. Our screening revealed a large variability of the enzyme activity in individuals with similar electrophysiological responses. In some moths the sensillar esterase was not detectable, i.e. present with 100-fold less activity. However, such variable esterase activity showed no correlation to the time course of the receptor potential. Thus, enzymatic pheromone degradation does not seem to be involved in the rapid pheromone inactivation at the end of the stimulus, but rather serves as the final pheromone sequestration step.

Identification of cytoskeletal proteins in the antennae of the silkmoths Antheraea polyphemus and A. pernyi

Although the overall cytoskeletal morphology of the olfactory dendrite in the antennae of the silkmoths Antheraea polyphemus and A. pernyi is known, the cytoskeleton proteins that structurally and functionally support these structures remain to be identified in this paper, we describe the identification of tubulin, actin and intermediate filament-like proteins in the olfactory dendrites, and motor proteins such as kinesin and unconventional myosin in the antennal branches by the use of antibodies. We also show that the tubulins within the olfactory dendrites and in the antennal branches are acetylated. This study provides valuable information concerning the possible role of these proteins in transduction, transport and motility, as is evident in other systems.