A modified method to analyse cell proliferation using EdU labelling in large insect brains

Abstract

The study of neurogenesis is critical to understanding of the evolution of nervous systems. Within invertebrates, this process has been extensively studied in Drosophila melanogaster, which is the predominant model thanks to the availability of advanced genetic tools. However, insect nervous systems are extremely diverse, and by studying a range of taxa we can gain additional information about how nervous systems and their development evolve. One example of the high diversity of insect nervous system diversity is provided by the mushroom bodies. Mushroom bodies have critical roles in learning and memory and vary dramatically across species in relative size and the type(s) of sensory information they process. Heliconiini butterflies provide a useful snapshot of this diversity within a closely related clade. Within Heliconiini, the genus Heliconius contains species where mushroom bodies are 3-4 times larger than other closely related genera, relative to the rest of the brain. This variation in size is largely explained by increases in the number of Kenyon cells, the intrinsic neurons which form the mushroom body. Hence, variation in mushroom body size is the product of changes in cell proliferation during Kenyon cell neurogenesis. Studying this variation requires adapting labelling techniques for use in less commonly studied organisms, as methods developed for common laboratory insects often do not work. Here, we present a modified protocol for EdU staining to examine neurogenesis in large-brained insects, using Heliconiini butterflies as our primary case, but also demonstrating applicability to cockroaches, another large-brained insect.

Fig 1. Different ways of incorporating EdU that were trialed during our protocol development.

From top to bottom: injection into the pupae, painting EdU onto plant material in larvae, and brain and whole pupa incubations.

Fig 2. Positive EdU staining in Drosophila melanogaster (prepupa, control).

A and C, Schematic drawings of the brain of the Drosophila larvae. B, EdU staining in the central brain. D, EdU staining shown in higher magnification for one lobe. Scale bars = 200 μm in B, 50 μm in D.

Fig 3. Nuclei stained by EdU in the optic lobe of Dryas iulia young pupa (Day 1).

A, Nuclei staining with Hoechst 3342 (magenta). B, EdU staining (green). C, EdU and nuclei double staining. Scale bars = 20 μm.

Fig 4. Positive EdU staining in the mushroom body of Dryas iulia young pupae (Day 1), with lower numbers of EdU⁺ cells with shorter incubations.

A, Schematic drawings of the brain of butterflies and location of the staining shown. Nuclei staining with Hoechst 33342 (magenta) after 90’ (B) and 40’ incubation period (E). EdU staining (green) after a 90’ (C) and 40’ incubation period (F). EdU and nuclei double staining after a 90’ (D) and 40’ incubation period (G). Scale bars = 50 μm.

Fig 5. EdU positive staining in the mushroom body of another large-brained insects, Diploptera punctata (4 weeks old).

A, Schematic drawings of the brain showing the location of the imaged cells. B, E, nuclei staining with Hoechst 33342 (magenta). C, F, EdU staining (green). D, G, EdU and nuclei double staining. The arrows indicate EdU⁺ cells. MB: Mushroom body, Ca: Calyx, KCs: Kenyon cells. Scale bars = 100 μm.

Fig 6. Compatibility of EdU and immunostainings in one hemisphere of the brain of a Dryas iulia larva (5^th instar).

Triple staining of Hoechst 33342, EdU and anti-HRP (horseradish peroxidase) in Dryas iulia late larva (5^th instar). A, Schematic drawings of the brain showing the location of the imaged cells. B, F nuclei staining with Hoechst 33342 (magenta). C, G EdU staining (green). D, H, HRP staining (yellow). E, I, triple staining. Scale bars = 100 μm in B-E, 50 μm in F-I.