Adult neurogenesis does not explain the extensive post-eclosion growth ofHeliconiusmushroom bodies

Abstract

Among butterflies, Heliconius have a unique behavioural profile, being the sole genus to actively feed on pollen. Heliconius learn the location of pollen resources, and have enhanced visual memories and expanded mushroom bodies, an insect learning and memory centre, relative to related genera. These structures also show extensive post-eclosion growth and developmental sensitivity to environmental conditions. However, whether this reflects plasticity in neurite growth, or an extension of neurogenesis into the adult stage, is unknown. Adult neurogenesis has been described in some Lepidoptera, and could provide one route to the increased neuron number observed in Heliconius. Here, we compare volumetric changes in the mushroom bodies of freshly eclosed and aged Heliconius erato and Dryas iulia , and estimate the number of intrinsic mushroom body neurons using a new and validated automated method to count nuclei. Despite extensive volumetric variation associated with age, our data show that neuron number is remarkably constant in both species, suggesting a lack of adult neurogenesis in the mushroom bodies. We support this conclusion with assays of mitotic cells, which reveal very low levels of post-eclosion cell division. Our analyses provide an insight into the evolution of neural plasticity, and can serve as a basis for continued exploration of the potential mechanisms behind brain development and maturation.

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

“… 18 In honeybees 32 and leaf-cutting ants, 33 however, olfactory learning has been linked to increased synapse density without expansion of the calyx. In addition, we extend previous reports of an absence of adult neurogenesis in Heliconiini, 64 by showing that learning experience does not promote an increase in Kenyon cell number ( Figure 3 ; Table S11 ), as observed in some hemimetabolous insects. 65 , 66 Differences in synapse count between Heliconius erato groups therefore appear to be a result of changes in the number of synapses per Kenyon cell ( Table S12 ).…”

“…Cell numbers within each box were automatically counted using the Modular Image Analysis (MIA) and Stardist plugins in ImageJ. 64 , 71 , 72 Stardist detects objects with star-convex shape priors and can be used for detecting cells. Total cell counts were then estimated by multiplying the average density by the total volume of the cell cluster.…”

Mosaic evolution of a learning and memory circuit in Heliconiini butterflies

“…This provided the clearest view of the mushroom body lobes, which undergo significant post-eclosion growth, further obscuring anatomical boundaries [31]. As post-eclosion growth does not involve adult neurogenesis [70], the morphology of the young adult lobes will reliably reflect variation in KC sub-types. Nevertheless, we tested for age effects (S3 Fig), using Dryas iulia and Heliconius erato aged to between 9-10 days old, at which point they are sexually mature, to assess for differential effects of age on relative lobe size (see statistical analysis).…”