Enhanced long-term memory and increased mushroom body plasticity in Heliconius butterflies

As highly labile structures on both individual and evolutionary time-scales, the mushroom bodies, a key site of learning and memory in insects, are an excellent model for investigating the evolution of cognitive variation. We explored the behavioural consequences of mushroom body expansion in Heliconius butterflies, which possess greatly expanded mushroom bodies relative to their closest outgroups. We conducted long-term visual memory assays across three Heliconius and three other Heliconiini species using trained food-colour associations. We confirm robust differences between clades, with Heliconius exhibiting greater fidelity to the trained colour after 8 days without reinforcement compared to other Heliconiini, with further evidence of stable preferences at 13 days. We extended this analysis to consider the plastic response of the mushroom body calyces over this time period, measuring the volume of the mushroom body calyx, and the number of neurons and synapses it contains. We find substantial post-eclosion expansion and synaptic pruning in calyx of Heliconius erato, but not in Dryas iulia. In Heliconius erato, visual associative learning experience specifically is associated with a greater retention of calyceal synapses. At an individual level, fidelity to the trained colour in Heliconius erato was also positively correlated with synapse number. These results point to an enhanced visual long-term memory across Heliconius, facilitated not only by phylogenetic expansion of the mushroom body, but also changes in its developmental response to learning experience. The co-evolution of mushroom body expansion, plasticity and specific behaviours provides an important case study in the evolution of cognition. Significance StatementHow are cognitive differences between species supported by evolutionary changes in the brain? We investigated this question using Heliconius butterflies which have expanded mushroom bodies, a region of the insect brain involved in learning and memory. We show that Heliconius have more stable visual long-term memories and exhibit more substantial age- and experience-related plasticity than a closely related genus with smaller mushroom bodies. Recall accuracy was also predicted by synapse number in Heliconius erato, but not Dryas iulia, suggesting functional importance. These results suggest that increases in the size of specific brain regions and changes in their plastic response to experience may co-evolve to shape the evolution of cognition.