Pheromone-producing cells in the silkmoth, Bombyx mori: identification andtheir 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 i nto two distinct layers, the outer and inner layers, by enzymatic digestion with papain. The outer layer mainly consists of cuticle, while the inner l ayer consists of homogeneous cells with many refractile granules. The solub ilized microsome fraction prepared from the inner layer retained the abilit y to produce bombykol in vitro, whereas the outer layer fraction did not pr oduce bombykol. Moreover, in tissue incubations, the inner layer - but not the outer layer - produced bombykol in response to the pheromonotropic pept ide TKYFSPRLamide, ionomycin and calcium ionophore A23187. These results in dicate that the inner-layer cells are indeed the pheromone-producing cells, which retain their functional integrity after separation with papain. Thes e cells could be cultured successfully in Grace's medium for at least 5 day s. The presence or absence of pheromonotropic stimuli prior to dissection grea tly influenced the size, number and distribution of refractile granules in the cytoplasm of the pheromone-producing cells. Staining with Nile Red prov ed that these refractile granules were lipid droplets. When pheromone produ ction was studied under normal conditions or stimulated in decapitated fema les with pheromone-biosynthesis-activating neuorpeptide (PBAN) charge, the size of lipid droplets observed in the pheromone-producing cells reduced pr ominently and their number increased dramatically with time. By contrast, w hen pheromone production was suppressed by decapitation, the size and numbe r of the lipid droplets remained constant. Lipid droplets observed in the p heromone-producing cells could be carriers of pheromone precursors and/or t he pheromone bombykol. The present results suggest that the isolated cell p reparation can be used for quantitative visualization of the cellular dynam ics during pheromone production in B. moll. (C) 2000 Elsevier Science Ltd. All rights reserved.