Dissections of Larval, Pupal and Adult Butterfly Brains for Immunostaining and Molecular Analysis

Butterflies possess impressive cognitive abilities, and investigations into the neural mechanisms underlying these abilities are increasingly being conducted. Exploring butterfly neurobiology may require the isolation of larval, pupal, and/or adult brains for further molecular and histological experiments. This procedure has been largely described in the fruit fly, but a detailed description of butterfly brain dissections is still lacking. Here, we provide a detailed written and video protocol for the removal of Bicyclus anynana adult, pupal, and larval brains. This species is gradually becoming a popular model because it uses a large set of sensory modalities, displays plastic and hormonally controlled courtship behaviour, and learns visual mate preference and olfactory preferences that can be passed on to its offspring. The extracted brain can be used for downstream analyses, such as immunostaining, DNA or RNA extraction, and the procedure can be easily adapted to other lepidopteran species and life stages.