Abstract
The colorful wings of butterflies are emerging as model systems for evolutionary and developmental studies. Some of these studies focus on localizing gene transcripts and proteins in wings at the larval and pupal stages using techniques such as immunostaining and in situ hybridization. Other studies quantify mRNA expression levels or identify regions of open chromatin that are bound by proteins at different stages of wing development. All these techniques require dissection of the wings from the animal but a detailed video protocol describing this procedure has not been available until now. Here, we present a written and accompanying video protocol where we describe the tools and the method we use to remove the larval and pupal wings of the African Squinting Bush Brown butterfly Bicyclus anynana. This protocol should be easy to adapt to other species.
Highlights
Multiple studies on butterflies are focused on understanding the evolutionary and developmental genetics of their colorful wing patterns
Examples include the discovery of the involvement of the gene optix in wing pattern mimicry of Heliconius butterflies [1,2]; discovery of doublesex as a mimicry supergene in Papilio polytes [3]; involvement of Wnt signaling in wing pattern of butterflies such as Junonia coenia, Heliconius erato and Vanesssa cardui [4,5,6]; involvement of the genes spalt and BarH-1 in the wing pigmentation of multiple Pieris and Colias species [7,8,9]; and the involvement of calcium signaling in wing patterning in Junonia orithya [10] to mention a few
Butterflies are becoming a model system to understand the process of color pattern formation in Biology
Summary
Multiple studies on butterflies are focused on understanding the evolutionary and developmental genetics of their colorful wing patterns. Gene expression and RNAi studies have proposed that many wound healing network genes are expressed in eyespots [23], that a positional-information mechanism is involved in the formation of the concentric rings [16,20], and that a reaction-diffusion mechanism is involved in setting up the eyespot centers [22] using gene expression and functional analysis via CRISPR-Cas9 [24,25] All these studies require the removal of wings from the bodies of larvae and/or pupae to examine patterns of gene expression. We describe the process of larval and pupal wing removal using a video and explain the process along with all the tools and chemicals needed in the main text below This protocol can supplement other similar experiments such as live cell imaging in vivo used to understand cell differentiation and dynamics [28,29]
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