Live Cell Imaging of Butterfly Pupal and Larval Wings In Vivo

Abstract

Butterfly wing color patterns are determined during the late larval and early pupal stages. Characterization of wing epithelial cells at these stages is thus critical to understand how wing structures, including color patterns, are determined. Previously, we successfully recorded real-time in vivo images of developing butterfly wings over time at the tissue level. In this study, we employed similar in vivo fluorescent imaging techniques to visualize developing wing epithelial cells in the late larval and early pupal stages 1 hour post-pupation. Both larval and pupal epithelial cells were rich in mitochondria and intracellular networks of endoplasmic reticulum, suggesting high metabolic activities, likely in preparation for cellular division, polyploidization, and differentiation. Larval epithelial cells in the wing imaginal disk were relatively large horizontally and tightly packed, whereas pupal epithelial cells were smaller and relatively loosely packed. Furthermore, larval cells were flat, whereas pupal cells were vertically elongated as deep as 130 μm. In pupal cells, many endosome-like or autophagosome-like structures were present in the cellular periphery down to approximately 10 μm in depth, and extensive epidermal feet or filopodia-like processes were observed a few micrometers deep from the cellular surface. Cells were clustered or bundled from approximately 50 μm in depth to deeper levels. From 60 μm to 80 μm in depth, horizontal connections between these clusters were observed. The prospective eyespot and marginal focus areas were resistant to fluorescent dyes, likely because of their non-flat cone-like structures with a relatively thick cuticle. These in vivo images provide important information with which to understand processes of epithelial cell differentiation and color pattern determination in butterfly wings.

Self-Similarity, Distortion Waves, and the Essence of Morphogenesis: A Generalized View of Color Pattern Formation in Butterfly Wings

“…These spots and marks are likely produced by organizing cells for adult eyespots. The epithelial distortion structures of the prospective elements have also been confirmed by in vivo imaging of the living tissue (Ohno and Otaki 2015a;Iwasaki et al 2017). The association of the organizing centers with distortion structures may be called the distortion rule for organizing centers.…”

Spatial patterns of correlated scale size and scale color in relation to color pattern elements in butterfly wings

“…Except E. kühniella and M. sexta, polyploidy has not been demonstrated in lepidopteran wings. However, we have already reported emergence of large cells with large nuclei in pupal developing wings in J. orithya in vivo (Ohno and Otaki, 2015a). Thus, we believe that polyploidization could be widespread in butterflies and moths.…”

“…Morphogenic signals are released laterally to form the secondary organizing centers, which repeat genome amplification and the scale-building process to form PFE (parafocal element) and eyespot black ring. may be physically connected with each other with epidermal feet and with cytonemes or nanotubes, forming a cluster of cells (Ohno and Otaki, 2015a) to regulate the total number of genomic amplifications.…”

“…From this perspective, the characterization of scale cell development is required to understand the nature of the putative morphogenic signal. Although scale cell development has been observed in some studies (Nardi and MageeAdams, 1986;Cho and Nijhout, 2013;Iwata et al, 2014;Ohno and Otaki, 2015a), basic information on developmental morphological changes remains scarce, and exact relationships and interactions between scale size and scale color have been reported only in J. orithya and partially in J. oenone (Kusaba and Otaki, 2009).…”

Synergistic Damage Response of the Double-Focus Eyespot in the Hindwing of the Peacock Pansy Butterfly

“…Although there are some classical histological studies on developing wing tissues of butterflies and moths [34,43,44], real-time live imaging studies have just begun on developing wing tissues and organizing cells [30,37,45]. The distortion hypothesis should be tested in the future in light of the importance of mechanical forces in development [46,47].…”