Mapping gene expression in whole larval brains of Bicyclus anynana butterflies

Butterfly larvae display intricate cognitive capacities and behaviors but relatively little is known about how those behaviors alter their brains at the molecular level. Here we optimized a hybridization chain reaction 3.0 (HCR v3.0) protocol to visualize the expression of multiple RNA molecules in fixed larval brains of the African butterfly Bicyclus anynana. We optimized the polyacrylamide gel mounting, fixation, and sample permeabilization steps, and mapped the expression domains of ten genes in whole larval brain tissue at single-cell resolution. The genes included optomotor blind (omb), yellow-like, zinc finger protein SNAI2-like (SNAI2), weary (wry), extradenticle (exd), Synapsin, Distal-less (Dll), bric-à-brac 1 (bab1), dachshund (dac), and acetyl coenzyme A acetyltransferase B (AcatB). This method can be used alongside single-cell sequencing to visualize the spatial location of brain cells that change in gene expression or splicing patterns in response to specific behaviors or cognitive experiences.