Live Cell Imaging of Butterfly Pupal and Larval Wings In Vivo

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.

. How to perform dye loading to a pupal wing tissue by the sandwich method.
Various small chemicals including many kinds of fluorescent dyes and inorganic salts may be loaded successfully, although we have not successfully transferred plasmid DNA. This sandwich method, which is based on the wing flipping method invented in 2009 for observing developmental changes in vivo [1], has already been utilized in the previous studies [2][3][4]. Furthermore, this wing flipping method has been applied to an electroporation method for gene transfer in Bombyx mori [5]. Readers are also encouraged to refer to these papers. Detailed steps are described below.
(1) A freshly pupated pupa is placed on a piece of plastic film (e.g., Parafilm M®) approximately 30 min after pupation. The tip of the right forewing is carefully picked up with forceps. To do so, recognize wing edges by naked eyes and do not pick up or damage other parts of the body.
(2) The right forewing is slowly picked up in a direction from peripheral to basal portions to expose the hindwing surface. The forewing should be flipped, preferably to the wing base.
(3) The flipped forewing is held vertically with a tip of the index finger. If the forewing is more angled, the wing base will bleed. Such an individual is not usable anymore.
(4) Approximately 20 μL of chemical solution (e.g., fluorescent dye solution for confocal imaging studies) is applied onto the exposed hindwing surface using a pipette. The forewing should be kept in the vertical position during this step.
(5) The vertically held forewing is now placed back onto the solution. The chemical solution is now sandwiched between the fore-and hindwings (see Figure 1a). If the sandwiched chemical solution is not enough, additional 20 μL can be applied from the side at this point, resulting in a total volume of approximately 40 μL. This volume is the maximum to be held between the wings. Then, the whole system is placed in a simple humidified chamber to let chemicals penetrate to wing epithelial cells (see (8) Insect Ringer's solution is applied again to wash out chemicals completely.
(9) Liquid spilled over is wiped out by a piece of paper towel (e.g., Kimwipes TM ).
Approximately 10 μL of insect Ringer's solution is placed onto the hindwing surface.
Without this, the forewing would directly stick onto a piece of slide glass in the next step, leading to wrinkle formation and bubble trapping. These in turn may potentially cause artefacts in subsequent observations.
(10) A piece of slide glass is placed onto the hindwing. The plastic film and slide glass are then held together by your fingertips at the circled position.
(11) The whole system, held by your fingertips at the circled position, is now turned upside down. Do not touch the pupa at all.
(12) The plastic film is removed.
(13) Liquid on the slide glass is wiped out by a piece of folded paper towel. If this step is not performed, fluorescent images are not of high quality due to the liquid between the hindwing and slide glass. It is now ready to set the whole system on a stage of inverted microscope.