Dissection of Larval and Pupal Wings of Bicyclus anynana Butterflies

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.

Keywords: Bicyclus anynana; butterflies; wing dissection.

Figure 1

Dissection of larval and pupal wings of Bicyclus anynana. (A). Larval wings are located laterally (black arrows), dorsal to the second and third thoracic legs. The region for incision is marked by a dotted line. (B). A larval forewing is located dorsally to the second thoracic leg. (C). A larval hindwing is located beside the third thoracic leg. (D). Early larval wings can be identified by finding the white tissue (black arrow) around the thoracic legs. Furthermore, to release the pressure due to the gut it is recommended to make an initial dorsal incision (through which the gut can extend) before the lateral incision is made. (E). For the dissection of pupal wings make incision as marked by the dotted line. (F). Pupal forewing. (G). Pupal hindwing. (H). Early (16–26 h after pupation) pupal forewing.

Figure 2

Larval and Pupal wings of Bicyclus anynana. (A) Early larval forewing showing the prominent white tissue that will differentiate into the trachea. Wings should be handled in this region during dissections; (B) Early larval hindwing; (C) Late larval forewing; (D) Late larval hindwing; (E) Pupal forewing; (F) Pupal hindwing.