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

Abstract

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.

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

“…Growing scales have been observed at time points much later in this species: 45 h postpupation [ 49 ]. In other species, growing scales have been observed 20 h [ 44 ] and 36 h [ 31 ] postpupation, and the detailed time course of scale development has been studied [ 31 , 32 ].…”

“…We have previously reported how to visualize the wing epithelial tissue of the pale grass blue butterfly in vivo using a confocal microscope system [ 48 , 49 ]. FB28 and other fluorescent probes were topically applied to the wing epithelial tissue using the sandwich method as follows.…”

“…We therefore examined whether there were epithelial cells containing intracellular chitin. Taking advantage of the fact that pupal wing tissues are located on the surface of the pupal body, forewing-lift operations have been performed to directly observe live wing epithelial tissues and cells in vivo since 2009 [ 42 ] and have been used for bioimaging [ 43 , 44 , 45 , 46 , 47 , 48 , 49 ] and for studying color pattern changes [ 40 ]. In this study, using this method, FB28 and other fluorescent probes were applied, and stained live cells and their intracellular structures were visualized in vivo.…”