Surgical Manipulations on Pupal Wings from the African Butterfly Bicyclus anynana: Damage and Cauteries

INTRODUCTION

This protocol describes cautery experiments with young pupal wings of the African butterfly Bicyclus anynana to examine the potential role of groups of cells in wing pattern determination. Experiments can be performed on either the left or right wing, leaving the other wing as a control. The pupal wing case overlies a stack of four sheets of epithelial cells that will form, in turn, the dorsal and ventral surfaces of the forewing and then the respective surfaces of the hindwing. The dorsal surface of the forewing is immediately underneath the cuticle. The trachea or wing veins of the forewing are visible through the pupal cuticle under a binocular microscope, providing landmarks. Raised “bumps” or irregularities on the pupal cuticle indicate where the eyespot organizers (or foci) are present on the underlying forewing. The wild-type pattern of the dorsal forewing consists of a small anterior eyespot and a large posterior eyespot; each eyespot has a central white pupil, a black inner ring, and an outer gold ring. Experiments that involve a short insertion of sharpened needles through the pupal cuticle can explore the effects of damage to groups of cells at different times from shortly after pupation until pattern determination has occurred (~24 h after pupation). This type of experiment originally helped to characterize the eyespot organizers because early focal damage to the location of these cells can result in shrinkage or complete elimination of the corresponding eyespot in the adult wing (relative to the control wing).