Periodic Reporting for period 4 - MuBoEx (Mushroom Body Expansion in Heliconius butterflies)
Période du rapport: 2022-09-01 au 2024-01-31
How and why do some species evolve bigger brains than others? The major goal of our project is to understand the evolutionary and development mechanisms that control the expansion of brain components, and explore its behavioural consequences. We study the evolution of a pair of structures in the insect brain called the mushroom bodies. Mushroom bodies are prominent ‘higher order’ structures that integrate sensory information and store memories of past experience. Insect Mushroom bodies share a conserved ground plan, but their size and morphology vary extensively across species.
We have established a new study system, Heliconius butterflies, to explore the evolution of mushroom body diversity. Heliconius mushroom bodies are notably expanded and hypothesised to form the basis of enhanced spatial memory associated with a dietary innovation, whereby adult Heliconius collect and digest pollen grains to obtain an adult source of amino acids. Heliconius collect pollen from a restricted range of plants, apparently learning the locations of these resources. This cognitive adaptation is hypothesised to involve “some elaboration of the nervous system”, with the mushroom bodies being a key, but untested, candidate.
Our project aims to answer four key questions that span behaviour, neuroanatomy and development to provide a cohesive understanding of both how and why mushroom bodies expanded in Heliconius:
1. How does mushroom body expansion enhance cognitive ability?
2. What aspects of mushroom body structure contribute to its volumetric expansion?
3. What developmental mechanisms control mushroom body expansion?
4. What is the genetic basis of mushroom body expansion?
Our objectives tackle major questions that cut across study systems: Does brain expansion necessarily increase computational power? Does it reflect novel functions formed by modified connections between brain regions? How is brain size and structure regulated? How do environmental cues interact with developmental programs? By studying a tractable group of insects, which combine extreme trait differences with the ability to use a range of experimental techniques, we will provide new clues to these big questions.
We have established a new study system, Heliconius butterflies, to explore the evolution of mushroom body diversity. Heliconius mushroom bodies are notably expanded and hypothesised to form the basis of enhanced spatial memory associated with a dietary innovation, whereby adult Heliconius collect and digest pollen grains to obtain an adult source of amino acids. Heliconius collect pollen from a restricted range of plants, apparently learning the locations of these resources. This cognitive adaptation is hypothesised to involve “some elaboration of the nervous system”, with the mushroom bodies being a key, but untested, candidate.
Our project aims to answer four key questions that span behaviour, neuroanatomy and development to provide a cohesive understanding of both how and why mushroom bodies expanded in Heliconius:
1. How does mushroom body expansion enhance cognitive ability?
2. What aspects of mushroom body structure contribute to its volumetric expansion?
3. What developmental mechanisms control mushroom body expansion?
4. What is the genetic basis of mushroom body expansion?
Our objectives tackle major questions that cut across study systems: Does brain expansion necessarily increase computational power? Does it reflect novel functions formed by modified connections between brain regions? How is brain size and structure regulated? How do environmental cues interact with developmental programs? By studying a tractable group of insects, which combine extreme trait differences with the ability to use a range of experimental techniques, we will provide new clues to these big questions.
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.
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.
This project has developed an insect system to link neural and cognitive evolution. We have shown that Heliconius mushroom bodies (insect learning and memory centers) are among the largest of any insect, and 4-fold larger than typical for Lepidoptera including their closest relatives. We have developed this system as an integrative model of brain expansion, with studies spanning cognitive ecology, through to densely sampled comparative genomics, and comparative neuroanatomy spanning cellular variation to projection pathways. This work includes some of the most comprehensive analyses of neural adaptations in insects to date, combining phylogenetically broad sampling with precise quantitative neural measurements. Our behavioural experiments have demonstrated learning and memory enhancements in specific contexts in Heliconius, including long term memory and combinatorial learning. We have also provided the first experimental evidence of site fidelity and spatial learning in any Lepidoptera. Our genomic and developmental work has combined a range of molecular approaches to produce major datasets which are providing unmatched views into the selection pressure shaping genome evolution and gene regulation across an insect radiation. In doing so they are revealing some of the first clues as to how mushroom body expansion is controlled and a genetic and cellular level. This work is establishing a major new system for comparative neurobiology which integrates data across multiple biological levels, and the divergent research focuses of vertebrate and invertebrate evolutionary neurobiology.