Real-Time In Vivo Imaging of the Developing Pupal Wing Tissues in the Pale Grass Blue Butterfly Zizeeria maha: Establishing the Lycaenid System for Multiscale Bioimaging

Abstract

To systematically analyze biological changes with spatiotemporal dynamics, it is important to establish a system that is amenable for real-time in vivo imaging at various size levels. Herein, we focused on the developing pupal wing tissues in the pale grass blue butterfly, Zizeeria maha, as a system of choice for a systematic multiscale approach in vivo in real time. We showed that the entire pupal wing could be monitored throughout development using a high-resolution bright-field time-lapse imaging system under the forewing-lift configuration; we recorded detailed dynamics of the dorsal and ventral epithelia that behaved independently for peripheral adjustment. We also monitored changes in the dorsal hindwing at the compartmental level and directly observed evaginating scale buds. We also employed a confocal laser microscopy system with multiple fluorescent dyes for three-dimensional observations at the tissue and cellular levels. We discovered extensive cellular clusters that may be functionally important as a unit of cellular communication and differentiation. We also identified epithelial discal and marginal dents that may function during development. Together, this lycaenid forewing system established a foundation to study the differentiation process of epithelial cells and can be used to study biophysically challenging mechanisms such as the determination of color patterns and scale nanoarchitecture at the multiscale levels.