Socket Array Irregularities and Wing Membrane Distortions at the Eyespot Foci of Butterfly Wings Suggest Mechanical Signals for Color Pattern Determination.

Eyespot foci on butterfly wings function as organizers of eyespot color patterns during development. Despite their importance, focal structures have not been examined in detail. Here, we microscopically examined scales, sockets, and the wing membrane in the butterfly eyespot foci of both expanded and unexpanded wings using the Blue Pansy butterfly Junonia orithya. Images from a high-resolution light microscope revealed that, although not always, eyespot foci had scales with disordered planar polarity. Scanning electron microscopy (SEM) images after scale removal revealed that the sockets were irregularly positioned and that the wing membrane was physically distorted as if the focal site were mechanically squeezed from the surroundings. Focal areas without eyespots also had socket array irregularities, but less frequently and less severely. Physical damage in the background area induced ectopic patterns with socket array irregularities and wing membrane distortions, similar to natural eyespot foci. These results suggest that either the process of determining an eyespot focus or the function of an eyespot organizer may be associated with wing-wide mechanics that physically disrupt socket cells, scale cells, and the wing membrane, supporting the physical distortion hypothesis of the induction model for color pattern determination in butterfly wings.

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.

Protein Delivery to Insect Epithelial Cells In Vivo: Potential Application to Functional Molecular Analysis of Proteins in Butterfly Wing Development.

Protein delivery to cells in vivo has great potential for the functional analysis of proteins in nonmodel organisms. In this study, using the butterfly wing system, we investigated a method of protein delivery to insect epithelial cells that allows for easy access, treatment, and observation in real time in vivo. Topical and systemic applications (called the sandwich and injection methods, respectively) were tested. In both methods, green/orange fluorescent proteins (GFP/OFP) were naturally incorporated into intracellular vesicles and occasionally into the cytosol from the apical surface without any delivery reagent. However, the antibodies were not delivered by the sandwich method at all, and were delivered only into vesicles by the injection method. A membrane-lytic peptide, L17E, appeared to slightly improve the delivery of GFP/OFP and antibodies. A novel peptide reagent, ProteoCarry, successfully promoted the delivery of both GFP/OFP and antibodies into the cytosol via both the sandwich and injection methods. These protein delivery results will provide opportunities for the functional molecular analysis of proteins in butterfly wing development, and may offer a new way to deliver proteins into target cells in vivo in nonmodel organisms.

Butterfly Wing Color Pattern Modification Inducers May Act on Chitin in the Apical Extracellular Site: Implications in Morphogenic Signals for Color Pattern Determination.

Butterfly wing color patterns are modified by various treatments, such as temperature shock, injection of chemical inducers, and covering materials on pupal wing tissue. Their mechanisms of action have been enigmatic. Here, we investigated the mechanisms of color pattern modifications usingthe blue pansy butterfly Junoniaorithya. We hypothesized that these modification-inducing treatments act on the pupal cuticle or extracellular matrix (ECM). Mechanical load tests revealed that pupae treated with cold shock or chemical inducers were significantly less rigid, suggesting that these treatments made cuticle formation less efficient. A known chitin inhibitor, FB28 (fluorescent brightener 28), was discovered to efficiently induce modifications. Taking advantage of its fluorescent character, fluorescent signals from FB28 were observed in live pupae in vivo from the apical extracellular side and were concentrated at the pupal cuticle focal spots immediately above the eyespot organizing centers. It was shown that chemical modification inducers and covering materials worked additively. Taken together, various modification-inducing treatments likely act extracellularly on chitin or other polysaccharides to inhibit pupal cuticle formation or ECM function, which probably causes retardation of morphogenic signals. It is likely that an interactive ECM is required for morphogenic signals for color pattern determination to travel long distances.