We have pusured our four objectives in tandem, with dedicated, specialist team members working on each problem. We have made significant progress on each aim:
1. How does mushroom body expansion enhance cognitive ability?
To understand the evolutionary history and ecological associations driving mushroom body expansion we collected, stained and imaged in excess of 400 individual butterflies, representing ~45 species spanning the Heliconiini tribe. We used volumetric data from these images to reconstruct the evolution of mushroom body expansion and test whether it has a single origin that co-occurred with the origin of pollen feeding. We developed numerous cognitive assays to understand how Heliconius use information from their environment, and how this differs from other Heliconiini. These include tests of traits that must contribute to trap-line foraging, including spatial learning, landmark learning, circadian memory, reversal learning and long-term memory retention.
2. What aspects of mushroom body structure contribute to its volumetric expansion?
To go beyond volumetric comparisons we investigated what cellular changes cause differences in mushroom body size. We have traced projection pathways, which carry sensory information to the mushroom body, to show that mushroom body expansion is associated with increased visual specialisation. We have reconstructed wider neural pathways to show that this occurs in the context of more widely conserved neuromorphologies. We have also estimated how mushroom body size is determined by the number of intrinsic neurons, the Kenyon cells, and we have shown that although the density of connections between these neurons is conserved across species, they are more plastic in Heliconius, suggesting greater environmental sensitivity. Finally, we have traced sub-populations of Kenyon cells to reveal mosaic expansion of different cell groups, and have also identified smaller expansions in related cell types which interact with the mushroom body.
3. What developmental mechanisms control mushroom body expansion?
To understand how changes in mushroom body size are produced by deviation in brain development, we have sampled over 12 developmental stages that span the larval period, metamorphosis during pupation, and emergence of the adult butterfly. We have compared patterns of development and neural proliferation in two Heliconius with expanded mushroom bodies and two related Heliconiini with smaller MBs. We have developed protocols to image neural proliferation and measure rates of neurogenesis. We have identified key stages where mushroom body development diverges across species through the production of derived intermediate progenitor cells in Heliconius, and are now investigating how this is brought about by changes in gene regulation.
4. What is the genetic basis of mushroom body expansion?
Finally, to complement our developmental work, we have compiled a genomic dataset including 58 species from across the Heliconiini. This includes new genomes sequenced by our team, to have representatives of all Heliconiini genera, and re-assembled and improved genomes for many other species. We are used used this dataset to identify protein coding genes and regulatory elements whose patterns of molecular evolution suggest a role in mushroom body evolution. We have collected data on gene expression and open chromatin profiles across development to identify changes in gene regulation associated with altered developmental trajectories.
