Pheromone-producing cells in the silkmoth, Bombyx mori: identification and their morphological changes in response to pheromonotropic stimuli.

A method to isolate functional clusters of viable pheromone gland cells of Bombyx mori was developed. The 8th-9th intersegmental invaginated membrane corresponding to the pheromone gland was dissected, trimmed and separated into two distinct layers, the outer and inner layers, by enzymatic digestion with papain. The outer layer mainly consists of cuticle, while the inner layer consists of homogeneous cells with many refractile granules. The solubilized microsome fraction prepared from the inner layer retained the ability to produce bombykol in vitro, whereas the outer layer fraction did not produce bombykol. Moreover, in tissue incubations, the inner layer - but not the outer layer - produced bombykol in response to the pheromonotropic peptide TKYFSPRLamide, ionomycin and calcium ionophore A23187. These results indicate that the inner-layer cells are indeed the pheromone-producing cells, which retain their functional integrity after separation with papain. These cells could be cultured successfully in Grace's medium for at least 5days.The presence or absence of pheromonotropic stimuli prior to dissection greatly influenced the size, number and distribution of refractile granules in the cytoplasm of the pheromone-producing cells. Staining with Nile Red proved that these refractile granules were lipid droplets. When pheromone production was studied under normal conditions or stimulated in decapitated females with pheromone-biosynthesis-activating neuorpeptide (PBAN) charge, the size of lipid droplets observed in the pheromone-producing cells reduced prominently and their number increased dramatically with time. By contrast, when pheromone production was suppressed by decapitation, the size and number of the lipid droplets remained constant. Lipid droplets observed in the pheromone-producing cells could be carriers of pheromone precursors and/or the pheromone bombykol. The present results suggest that the isolated cell preparation can be used for quantitative visualization of the cellular dynamics during pheromone production in B. mori.