Butterfly pupal wing tissue with an eyespot organizer

Butterfly wing eyespots are developmentally determined at the early pupal stage, when prospective eyespot focal cells underneath the pupal cuticle focal spot function as eyespot organizers in the pupal wing tissue. Here, we performed light microscopy and transmission electron microscopy (TEM) to describe cellular structures of pupal wing tissue with an eyespot organizer immediately after pupation using the Blue Pansy butterfly Junonia orithya. The pupal forewing dorsal epidermis was a pseudostratified monolayer of vertically elongated epidermal cells. The apical portion of the cells adhered laterally to one another, but their medial and basal portions were thinner than the apical portion and were tilted to enclose cells at the center, forming a cellular cluster. The cellular cluster at the organizer was relatively large laterally and vertically. The apical portion of the cells and its corresponding cuticle at the organizer were thicker than those in the surroundings. The innermost cuticle layer was being synthesized, indicating high cuticle synthesis and secretion activity of the cells. At the medial and basal portions of the dorsal epidermis, there were many intracellular and extracellular vacuole-like globules, most likely containing extracellular matrix molecules. Some of the basal processes from epidermal cells extended to form protrusions of the basement membrane, which was often attended by hemocytes. These results suggest that the butterfly eyespot organizer is composed of a single or a few cellular clusters that secrete more cuticle than surrounding clusters, supporting the pupal cuticle hypothesis that cuticle formation is critical for eyespot color pattern determination in butterflies.