Spermathecal accessory glands from pupae ofTenebrio molitorwere cultured in vitro in Landureau S-20 medium with or without ecdysterone at a concentration of 5 μg/ml medium. Morphological changes were examined by electron microscopy. Tissue taken from pupae that have not been exposed to a peak of ecdysterone in vivo is only able to differentiate in medium with hormone, and then only partially, while tissue taken from pupae that have experienced an endogenous peak of ecdysterone is able to develop maximally irrespective of the presence or absence of hormone. The specific ultrastructural changes that occur in vitro correspond to those occurring in the gland in situ during the normal course of differentiation, and are: the formation of the pseudocilium, cell retraction and formation of an end apparatus, and cuticulogenesis in the ductule and main lumen of the gland. Pseudocilium formation does not appear to be ecdysterone-dependent, while cuticulogenesis requires ecdysterone for initiation of the process. Deposition of cuticle is an expression of an earlier commitment of the cells to this process, and is initiated by elevated levels of ecdysterone. Ecdysterone is not required for completion of cuticulogenesis
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