Characterization of neuronal apoptosis in the ventral ganglia of postembryonic silkworms Bombyx mori (Lepidoptera: Bombycidae)

A fluorescent triple staining method was developed to stain the cytoplasm of neurons red, the nuclei of all kinds of cells, including neurons, blue and the nuclei of apoptotic neurons in cyan in the twelve ventral ganglia (VG) of the Bombyx mori ventral nerve cord. This differential staining method was used to distinguish between apoptotic and normal neurons in the suboesophageal ganglion (SOG), thoracic ganglia (TG) 1 to TG3 and abdominal ganglia (AG) 1 to AG8 and also determine the changes in the numbers of apoptotic neurons that occur during postembryonic development. In most of the VG tested, neuronal apoptosis was most marked during the period from the end of larval life to the mid pupal stage. The greatest number of apoptotic neurons was found in SOG of day-5 pupae, TG1 to TG3 and AG1 to AG4 of day-1 pupae, and AG5 to AG8 of day-4 pupae. In vivo injection of 20-hydroxyecdysone (20E) into day-8 5th instar larvae resulted in both a considerable increase in the number of apoptotic neurons and cleavage of procaspase-3 into caspase-3, which induced neuronal apoptosis in SOG and AG6 to AG8 in day-1 pupae, and a slight increase in the number of apoptotic neurons in TG1. In TG3 and AG4, however, it had little effect on the number of apoptotic neurons or cleavage of procaspase-3. Treatment of the VG of both day-8 5th instar larvae and day-2 pupae with protein synthesis inhibitors by in vivo injection triggered a significant inhibition of neuronal apoptosis and procaspase-3 cleavage in most of these ganglia in day-1 pupae and day-4 pupae, but not TG3 and AG4, in which there was little procaspase-3 and caspase-3. In vivo injection of caspase-8 and -3 inhibitors into day-8 5th instar larvae and day-2 pupae led to a substantial inhibition of neuronal apoptosis and of procaspase-3 cleavage in SOG, AG6 and TAG, but not in TG3 or AG4 of day-1 pupae and day-4 pupae. These findings suggest that neurons that die in SOG, TG1 and AG6 to AG8 in day-1 and -4 pupae may undergo apoptosis induced by the synthesis of a new protein and caspase-8-and -3-implicated signal transduction by the increase in titre of 20E in the haemolymph but not the neuronal aopotosis in TG3 and AG4. This study provides neurobiologists with valuable information and a means of studying neuronal apoptosis in the nervous system of insects.