Live Detection of Intracellular Chitin in Butterfly Wing Epithelial Cells In Vivo Using Fluorescent Brightener 28: Implications for the Development of Scales and Color Patterns

Chitin is the major component of the extracellular cuticle and plays multiple roles in insects. In butterflies, chitin builds wing scales for structural colors. Here, we show that intracellular chitin in live cells can be detected in vivo with fluorescent brightener 28 (FB28), focusing on wing epithelial cells of the small lycaenid butterfly Zizeeria maha immediately after pupation. A relatively small number of cells at the apical surface of the epithelium were strongly FB28-positive in the cytosol and seemed to have extensive ER-Golgi networks, which may be specialized chitin-secreting cells. Some cells had FB28-positive tadpole-tail-like or rod-like structures relative to the nucleus. We detected FB28-positive hexagonal intracellular objects and their associated structures extending toward the apical end of the cell, which may be developing scale bases and shafts. We also observed FB28-positive fibrous intracellular structures extending toward the basal end. Many cells were FB28-negative in the cytosol, which contained FB28-positive dots or discs. The present data are crucial to understanding the differentiation of the butterfly wing epithelium, including scale formation and color pattern determination. The use of FB28 in probing intracellular chitin in live cells may be applicable to other insect systems.

bioimaging; butterfly wing; chitin; cuticle; epithelial cell; fluorescent brightener 28; fluorescent probe; in vivo imaging.