Fig 1.
Pupal wing cuticle patterns of 3 Junonia species.
All scale bars indicate 1 mm. (a) A whole J. orithya pupa. (b) A pupal wing surface. High magnification of (a). (c) Pupal cuticle focal spots and focal marks (focus-associated marks). They are labelled as first to sixth from the anterior to the posterior sides of a wing. (d) A pupal wing surface immediately before eclosion. The adult color pattern is seen through the pupal cuticle case, which demonstrates the correspondence between the pupal focal spots and adult eyespots. (e) A whole J. almana pupa. (f) A pupal wing surface. High magnification of (e). Focal spots and marks are observed. (g) A whole J. hedonia pupa. (h) A pupal wing surface. High magnification of (g). Focal spots are observed but there is no focal mark.
Fig 2.
Comparison of the pupal cuticle focal spots and adult eyespots in 3 Junonia species.
The relative focal spot (FS) volume is shown for 5 focal spots in a species. Mean values are shown and error bars indicate standard deviation. The relative focal spot volume shows that the second and fifth focal spots are larger than the other spots in J. orithya and J. almana, whereas all the spots have a similar size in J. hedonia. A similar pattern is seen in the adult wings of these species. In J. orithya and J. almana, the dorsal and ventral eyespot patterns are roughly similar to each other.
Fig 3.
Scatter plots showing the relationship between focal spot volume and eyespot area in J. orithya.
Five focal spots from 2 males and 2 females were examined, showing 20 points in total. Male and female spots are shown in green and pink, respectively. (a) Focal spot volume versus eyespot area. (b) Focal spot volume versus eyespot area with logarithmic scale.
Fig 4.
Sexual comparison of the size of adult eyespots and pupal focal spots.
(a) Adult major (fifth) eyespot. Highly significant differences are detected in width and area. (b) Pupal major (fifth) focal spot. No significant difference is detected in height, width, and volume of focal spots between sexes.
Fig 5.
3D structure of the pupal wing surface of J. orithya.
(a) A whole pupal wing surface. (b) A side view of a pupal wing surface. (c) A side view of a region of the major (fifth) focal spot. (d) Another side view of a region of the major (fifth) focal spot. (e) Size measurement of a focal spot. Diameter and height are approximately 200 μm and 20 μm, respectively. The black region is a focal mark (focus-associated mark), which is lower in height than its surroundings. Red lines and colored arrowheads in top and bottom panels indicate identical sites.
Fig 6.
3D structure of the underside of a J. orithya pupal case at a focal spot.
(a) An image of a pupal case upside down at a focal spot. (b) An obliquely positioned view. (c) A side view.
Fig 7.
Cross section of the pupal major (fifth) focal spot of J. orithya.
(a) An excised region of a focal spot. The non-stained image is at the top, and the toluidine blue-stained image is at the bottom. Sections were made along yellow lines from peripheral to basal regions. Section numbers are shown below the image. The epicuticle is stained by toluidine blue. Four regions are defined: focal spot (FS), focal mark (FM), and basal and peripheral regions. The thickness of the cuticle layer and the underneath cell layer were measured along the anterior, central, and posterior lines shown in pink. (b) Examples of sections. The autofluorescent image (top) is a combination of three (blue, green, and red) fluorescent images. A blue signal indicates the cuticle, whereas a green signal indicates the cell layer (wing tissue). In the two bright-field images (middle and bottom), the light brown layer is the pupal cuticle, and the light blue layer below is the cell layer (wing tissue). The epicuticle is also stained with blue on the surface of the cuticle. The section number is indicated at the top left-hand corner. (c) Three measurement points (anterior, central, and posterior) (top) and how to measure the cuticle layer thickness and cell layer thickness (bottom) using a VHX-2000 communication software (Keyence). A hollow is seen underneath the focal spot. (d) Cuticle thickness (top panel) and cell thickness (middle panel) along the central, anterior, and posterior lines. Raw data are plotted with dotted lines, and averaged data over 5 sections (the section at a given point and two sections before and after that point) are plotted with solid lines. The averaged data for the cuticle layer and cell layer along the central line are shown as relative values (a maximum point is adjusted to be 1.0; bottom panel), indicating a positional correspondence between the cuticle thickness and cell thickness. Below the three panels, the 4 regions of the sections are indicated.
Fig 8.
Scatter plot of the relationship between the cuticle thickness and cell thickness.
Three regions, i.e., focal spot (FS), focal mark (FM), and basal/peripheral regions, are indicated in different colors.
Fig 9.
3D structure of the eyespot of a J. orithya adult female.
Arrows indicate an eyespot focus. (a) A side view. (b) A side view with color scale. (c) Side view with expanded height scale (× 2). (d) An obliquely positioned view with expanded height scale (× 2).
Fig 10.
Height measurements of the eyespots of a J. orithya adult female.
Blue lines and colored arrowheads indicate identical sites in the top and bottom images of each panel. (a) A dorsal eyespot with color scale. (b) A ventral eyespot with color scale. Eyespots shown in (a) and (b) are on the opposite surface on the same wing. (c, d) Additional dorsal eyespots from 2 individuals.
Fig 11.
Height measurements of the dorsal eyespots of a J. orithya male.
Blue lines and colored arrowheads indicate identical sites in the top and bottom images of each panel. (a-c) Eyespots from 3 different individuals. (d) Eyespot region with scales removed. The eyespot region is circled.
Fig 12.
3D structure and height measurements of the dorsal eyespots of J. almana.
(a) A top-down view of the 3D structure. (b) An obliquely positioned view of the 3D structure. (c, d) Eyespots from 2 different individuals. Green lines and colored arrowheads indicate identical sites in the top and bottom images of each panel.