Dynamic morphological staging of Drosophila pupal development

This study provides a comprehensive analysis of the morphological-developmental characteristics of the pupal stage in Drosophila, a model organism for metamorphosis research. We have delineated the critical stages of pupal development, from the late third-instar larvae to adult emergence, through continuous photography and time-lapse imaging. Our findings include the identification of the head-movement phase and head-static phase as distinct transitional states preceding the pupal stage. We document pupal shell dynamics, including color transitions and spiracle development as staging markers, along with internal transformations like larval tracheal degeneration, mouthpart retraction, and the formation of the compound eyes, wings, appendages, and bristles. In conclusion, we have identified several critical periods: P12 serves as a definitive marker for the onset of segmentation during the pupal stage, P40-P50 represents an optimal period for observing the initiation of changes in eye and wing coloration, and P70 marks the beginning of the investigation into the morphological changes of the adult within the Drosophila pupal case. We also found that compared with Canton-S (CS) wild-type flies, dneurexin (dnrx) and dneuroligins (dnlgs) mutants showed some differences in development time and pupal tissue organization. These observations offer a detailed developmental map of the Drosophila pupal stage, which is essential for accurate experimental staging and sampling. This study's findings not only contribute to the fundamental understanding of insect metamorphosis but also provide a valuable resource for researchers utilizing Drosophila as a model system to study gene expression, development, and behavior.