Adult neurogenesis does not explain the extensive post-eclosion growth of Heliconius mushroom 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.

Keywords: Kenyon cells; neurodevelopment; plasticity; proliferation.

Figure 1.

Schematic drawing of the mushroom body (a) and segmentations of the Kenyon cell cluster (KCs, blue) and Calyx (CA, red) of Heliconius erato (b) and Dryas iulia (c). The small boxes of 25 µm × 25 µm × 15 µm represent an example of the sections counted. Scale bars: 200 µm.

Figure 2.

Age dependent variation in the volume of the mushroom body calyx (a) but not in Kenyon cell number (b). Boxes encompass the two middle quartiles with central line showing the median. Whiskers extend to furthest data point within 1.5 times the interquartile range. ***p < 0.001.

Figure 3.

Little evidence of adult neurogenesis in Heliconiini mushroom bodies. (a,a′), Schematic drawings of the brain of butterflies. Nuclei and EdU staining in Dryas iulia (b,d,f) and Heliconius erato (b′,d′,f′). Nuclei and pH3 staining in Dryas iulia (c,e,g) and Heliconius erato (c′,e′,g′). Scale bars = in (b,b′,c) and (c′); 200 µm in (d–f) and (d′–f′). White arrows indicate EdU + or pH3 + cells. Scale bars = 100 µm in (b,b′,c) and (c′); 200 µm in (d–g) and (d′–g′).

Figure 4.

Adult neurogenesis in the optic lobe (OL) of 0 days adult Heliconiini butterflies. (a). Parts of the optic lobe from Montgomery et al. [33]. (b,e) Nuclei staining. (c,f) EdU staining. (d,g) Nuclei and EdU double staining. LA: lamina, ME: medulla, AME: accessory medulla, LO: lobula, LOP: lobula plate, vLO: ventral lobe of the LO. B: Scale bars = 200 µm.