Developmental Changes in the Prothoracicotropic Hormone Content of the Bombyx mori Brain-Retrocerebral Complex and Hemolymph: Analysis by Immunogold Electron Microscopy, Quantitative Image Analysis, and Time-Resolved Fluoroimmunoassay

Abstract

The insect brain neuropeptide, prothoracicotropic hormone (PTTH), elicits the molting process by stimulating ecdysteroidogenesis in the prothoracic glands. Changes in the subcellular distribution of PTTH during the fifth larval instar and larval-pupal metamorphosis of Bombyx were investigated using immunogold electron microscopy and computerized image analysis. A time-resolved fluoroimmunoassay was employed to quantify the hemolymph PTTH titer and the hemolymph ecdysteroid titer was determined by radioimmunoassay. Immunoreactivity was apparent only in the cytoplasm of two pairs of dorsal-lateral neurosecretory cells (La) in the brain, the prothoracicotropes, and their axons that traverse the peripheral area of the brain neuropil and then enter the neurohemal lobes of the corpora cardiaca and end in the corpora allata. Immunospecificity was evident as noted by the restriction of the 5-nm gold particles to the neurosecretory granules. Synaptic contacts were noted frequently between the dendritic collaterals of the prothoracicotropes and neighboring collaterals from other neurons. The morphological data suggest that PTTH is synthesized and released throughout the fifth larval instar. The concentration of gold particles in the neurosecretory granules and the number of neurosecretory granules increased five times during the fifth instar, and these increases were correlated with surges in the hemolymph PTTH and ecdysteroid titers. The data suggest that PTTH biosynthesis, secretory granule formation, and secretion are three continuous and almost synchronized events.