Developmental dynamics of butterfly wings: real-time in vivo whole-wing imaging of twelve butterfly species

Abstract

Colour pattern development of butterfly wings has been studied from several different approaches. However, developmental changes in the pupal wing tissues have rarely been documented visually. In this study, we recorded real-time developmental changes of the pupal whole wings of 9 nymphalid, 2 lycaenid, and 1 pierid species in vivo, from immediately after pupation to eclosion, using the forewing-lift method. The developmental period was roughly divided into four sequential stages. At the very early stage, the wing tissue was transparent, but at the second stage, it became semi-transparent and showed dynamic peripheral adjustment and slow low-frequency contractions. At this stage, the wing peripheral portion diminished in size, but simultaneously, the ventral epithelium expanded in size. Likely because of scale growth, the wing tissue became deeply whitish at the second and third stages, followed by pigment deposition and structural colour expression at the fourth stage. Some red or yellow (light-colour) areas that emerged early were "overpainted" by expanding black areas, suggesting the coexistence of two morphogenic signals in some scale cells. The discal spot emerged first in some nymphalid species, as though it organised the entire development of colour patterns. These results indicated the dynamic wing developmental processes common in butterflies.

Figure 1

Developmental time course of the pupal forewing and hindwing of J. almana. The panel numbers indicate time points in min after the beginning of image recording within 1 h post-pupation. Different individuals are presented in a, b, and c. (a) Dynamics of a whole forewing and a hindwing. The red arrow in panel 1 indicates the possible front edge of the ventral epithelium. White arrows indicate possible boundary between the forewing proper and the peripheral region to be degraded. The proximal side of the major eyespot forms multiple bands (indicated as Multiband in the panel 8801) that are not seen in the final colour pattern. DS in the panel 8801 indicates the discal spot. CSS in the panel 9101 indicates the central symmetry system. See also Supplementary Video 1. (b) A hindwing. The numbers in the panel 8641 indicate the order of red emergence. The numbers in the panel 9341 indicate the order of black emergence. The black and yellow bands were numbered in the order of emergence from inside to outside. The Yellow #2 was overpainted entirely by the Black #2. See also Supplementary Video 2. (c) Another different hindwing with similar overpainting of multiple bands. A red area located at the distal portion of the major eyespot was also overpainted by the black area elongated from the central part of the major eyespot.

Figure 2

Peripheral adjustment of the pupal forewing of J. almana. This is the same individual as that in Fig. 1a. The panel numbers indicate time points in min after the beginning of image recording within 1 h post-pupation. See also Supplementary Video 2. The moving directions are shown in red arrows. Asterisks in the panels 3200 and 3350 indicate the peripheral portion to be degraded. Assuming that the pupal cuticle does not change in size, the gap distance between the edges of the pupal wing cuticle and the live pupal forewing were indicated by the vertical upward black arrows with U-type size indicators (Panels 3450–9000). The dorsal epithelium for the wing proper (DE), the ventral epithelium for the wing proper (VE), the border lacuna for the dorsal epithelium (BL(D)), the border lacuna for the ventral epithelium (BL(V)), extended trachea to the peripheral portion (E), and loosened trachea (L) are indicated.

Figure 3

Developmental time course of the pupal hindwing eyespot of J. orithya in the CuA₁ compartment. The panel numbers indicate time points in min after the beginning of image recording within 1 h post-pupation. (a) Colouration sequence of the eyespot development. A black arrow in the panel 8802 indicates the position of the eyespot focal white scales. Other black arrows indicate emergence of red or black areas. White arrows in the panel 9382 indicate the width of the focal white area, which diminishes in the following panels. (b) High magnification of the eyespot shown in a. The width of the focal white area is indicated by double-headed arrows, which diminish consecutively, showing an overpainting process. The boxed area is enlarged in c. (c) High magnification of the eyespot rings is shown in b. Upper double-headed arrows bridge two positional reference points (dusts on the cover film). The red-black boundary on the double-headed arrows is indicated by small black arrows. The positions of the boundary change in the next panels. Lower double-headed arrows indicate the width of the red ring, which diminishes in the next panels. These changes demonstrate the overpainting dynamics of colour pattern development.

Figure 4

Developmental time course of the pupal forewing and hindwing of V. indica. The panel numbers indicate time points in min after the beginning of image recording within 1 h post-pupation. (a) Time course of the forewing and hindwing development. DS indicates the discal spot emerged on the ventral forewing. See also Supplementary Video 3. (b) Higher magnification of a. Red emerged first, followed by black in the border symmetry system and its surroundings in the dorsal hindwing. Arrows indicate the positional reference point (a dust on the cover film), indicating the invasion of the black area into the red area. An asterisk indicates one of the black emerging points that may not belong to the border symmetry system.

Figure 5

Developmental time course of the pupal forewing of Argyreus hyperbius and Athyma selenophora. The panel numbers indicate time points in min after the beginning of image recording within 1 h post-pupation. (a) Argyreus hyperbius. Arrows in the panel 4001 indicate the edges of the dorsal and ventral epithelia. Arrows in the following panels indicate the forewing edge after the alignment of the dorsal and ventral epithelia. However, the edge was not aligned with the wing edge specified by the cuticle lines. DS in the panel 8001 indicates the discal spot. See also Supplementary Video 4. (b) Athyma selenophora. The cuticle of this pupae is metallic gold, and thus, some reflections cannot be avoided. In the panel 10201, the DS is indicated. In the panel 10501, the border symmetry system (BoSS) and the basal symmetry system (BSS) are shown. See also Supplementary Video 5.

Figure 6

Developmental time course of the pupal forewing of two closely related species, D. chrysippus and D. genutia. The panel numbers indicate time points in min after the beginning of image recording within 1 h post-pupation except b. (a) D. chrysippus. Red arrows in panels 9931 and 10001 indicate the areas that are soon invaded by the black expression. See also Supplementary Video 6. (b) A different individual of D. chrysippus. Red arrows in the panels 461 and 471 indicate the areas that are soon invaded by the black expression. See also Supplementary Video 7. (c) D. genutia. An arrow in the panel 9781 indicates an emerging vein-dependent black area that later expands to overwrite the surrounding red area. The vague black circles seen in panel 9001 and later in the wing area are not wing pigments but were often observed in this species. See also Supplementary Video 8.

Figure 7

Developmental time course of the pupal forewing and hindwing of Idea leuconoe and Ideopsis similis. The panel numbers indicate time points in min after the beginning of image recording within 1 h post-pupation except a and c. (a) Forewing of Idea leuconoe. Arrows indicate edges of the ventral epithelium. See also Supplementary Video 9. (b) Hindwing of Idea leuconoe. See also Supplementary Video 10. (c) Forewing of Ideopsis similis. See also Supplementary Video 11.

Figure 8

Developmental time course of the pupal forewing and hindwing of Z. maha. The panel numbers indicate time points in min after the beginning of image recording within 1 h post-pupation. (a) Ventral forewing and dorsal hindwing development of Z. maha argia (from Osaka). White arrows indicate moving edge of the dorsal hindwing epithelium. Red arrows indicate emerging black spots. See also Supplementary Video 12. (b) Ventral forewing colouration of Z. maha argia (from Osaka). Red arrows indicate emerging black spots. DS, discal spot. See also Supplementary Video 13. (c) Dorsal forewing development of Z. maha okinawana (from Okinawa) without operation. Black arrows indicate moving edge of the ventral epithelium. Blue arrows indicate newly defined forewing edge. Pink arrows indicate emerging black bands of the central symmetry system. Light blue arrow indicate structural colour. See also Supplementary Video 14.

Figure 9

Developmental time course of the pupal forewing and hindwing of L. phlaeas. The panel numbers indicate time points in min after the beginning of image recording within 1 h post-pupation except c. (a) Ventral forewing and dorsal hindwing development. White arrows indicate moving edge of the ventral hindwing epithelium. Blue arrows indicate moving edge of the dorsal hindwing epithelium. Pink arrow indicates retracted edge of the ventral hindwing epithelium in Panel 3001, after which the edges of the dorsal and ventral epithelia appeared to be aligned. Red and black arrows indicate emerging red and black areas, respectively. See also Supplementary Video 15. (b) Dorsal hindwing colouration. See also Supplementary Video 16. (c) Dorsal forewing development without operation. White and red arrows in Panel 6127 indicate emerging spots that constitute the central symmetry system. The red arrow indicates the discal spot. A green arrow in Panel 6227 indicates emerging marginal black area. The image recording was started approximately 39 h post-pupation, and this time point was the starting point of the panel numbers of this figure and corresponding Supplementary Video 17.

Figure 10

Developmental time course of the pupal forewing and hindwing of P. rapae. The panel numbers indicate time points in min after the beginning of image recording within 1 h post-pupation. (a) Ventral forewing and dorsal hindwing development. Pink arrows in Panels 5001 and 6001 indicate moving edge of the ventral hindwing epithelium. A blue arrow in Panel 5001 indicates moving edge of the dorsal hindwing epithelium. A yellow arrow indicates emerging yellow area. Black arrows indicate emerging black area. See also Supplementary Video 18. (b) Dorsal forewing development without operation. A blue arrow indicates emerging yellow area. Black arrows indicate emerging black area. The central black spots in the middle of the forewing did not seem to overpaint any yellow area. See also Supplementary Video 19.

Figure 11

Time table of pupal wing development. This time table is just a rough summary of the findings of the present study, assuming that the entire time span from pupation to eclosion is approximately 10,000 min (7 days). Some important events are also indicated based on the previous real-time imaging study using J. orithya.

Figure 12

Schematic diagram of the overpainting. In J. orithya, red areas emerge as patchy islands, but they merge into a single red area as they expand. At the same time, black areas emerge as patchy islands. They also merge into a single black area as they expand. As the black area further expands, the red area is overpainted by the black area. In this species, the focal white area is also overpainted by the surrounding black area (not shown in this diagram). In D. genutia, an expansion of the vein-dependent black area also involves the overpainting (not shown in this diagram). In L. phlaeas, black area emerges in the middle of the red area. In P. rapae, most yellow areas change to black areas. The central black spots emerge without any previous yellow colouration.