Fig. 4

Arcuate body and 3D reconstruction of protocerebral neuropils in E. spinosa. A 3D-reconstructed volume rendering of protocerebral sense organs and their connection to the brain, μCT scan, lateral view. Stippled lines indicate the positions of the sections shown in C. B Synapsin (SYN) immunolabeling for neuropil visualization in a whole-mount brain, dorsal view (blend mode). Note different labeling intensities of the two arcuate body layers. C Tubulin (TUB, green) and synapsin (magenta) immunolabeling with nuclear counterstain (NUC, gray), oblique optical sections from apical (top) to basal (bottom). Black arrowheads point to the antero-dorsal tract connecting the visual neuropil and arcuate body. Red arrowheads indicate horizontal neurite bundles projecting through the arcuate body. White arrowheads point to selected columnar neurites extending roughly perpendicular to the horizontal bundles. The white arrowhead highlights columnar neurites running on top of the arcuate body’s narrow midline portion. Asterisks mark neurite bundles (cross-sectioned) from antero-dorsal neurons that project into the brain neuropil. D 3D volume rendering of a tubulin- and synapsin-labeled brain, ventro-lateral, and dorso-lateral views (top and bottom, respectively). The brain centers were differently colored in the synapsin channel. Only relevant tubulin-labeled nerves/neurite bundles are shown (green). The black arrowhead marks the antero-dorsal tract between the visual neuropil and arcuate body. Yellow arrowheads mark the optic nerve branch targeting the antero-median neuropil. Red arrowheads point to the optic nerve branch entering the central brain neuropil. Black arrows point to the primary neurites of ventral neurons (top) contributing to the antero-median tract (bottom). Abbreviations: AB – arcuate body; AE – anterior eye; AMN – antero-median neuropil; AMT – antero-median tract; BR – brain; BRN – brain neuropil; DPL – dorso-posterior lobe; LO – lateral sense organ; ONV – optic nerve; PE – posterior eye; PNV – proboscis nerve; SVD – sub-visual domain; VN – visual neuropil