waves in pupal wing tissue. (a) An adult individual of Junonia orithya. (b) Dorsal hindwing of J. orithya. The two boxed areas (the basal region in the red box and the border region in the green box) correspond to the areas of analysis in the pupal wing. (c) The loading of a chemical solution (the pink arrow) into the pupal wing tissue via the sandwich method. (d, e) The pupal hindwing attached to a piece of cover glass at approximately 1 h after surgery. The green rectangular area (the border region) corresponds to the area shown in f and h. The pink rectangular area (the basal region) corresponds to the area shown in i and k. The red broken circle corresponds to the prospective eyespot area. The scale bar represents 1 mm. (f) Ca2+ signals in the M3 and CuA1 compartments (the border region). ROIs 1–8 were examined for intensity changes in g. The yellow arrow indicates the prospective eyespot area, which is darker than its surroundings. The red arrowheads indicate the wing veins. The scale bar represents 400 μm. See also Additional file 1: Movie S1. (g) Fluorescence intensity changes in 8 ROIs in arbitrary units (AUs) over time. (h) Propagating Ca2+ signals around the prospective eyespot area as indicated by the yellow arrow. The right and left panels (and f) show a single identical visual field with superimposed propagating signals. A shape of a wave at a given time point (in min) is depicted by a dotted circle. (i) Ca2+ signals around the discal cell (i.e., the basal region). Five ROIs are defined. The scale bar represents 400 μm. See also Additional files 2, 3: Movies S2, S3. (j) Fluorescence intensity changes in the 5 ROIs defined in i over time. (k) Another example of Ca2+ signals around the discal cell. The scale bar represents 400 μm. (l) Fluorescence intensity changes in the 4 ROIs defined in h over time.