Figure 1.Hierarchical nanostructure of wing scales in Parides eurimedes (Papilionidae). (A) Adult male with structurally-colored green patches on their dorsal forewings. Credit: iDigBio YPM Ent 433579. (B) SEM top-view and (C) FIB-SEM cross-sectional image of an adult green scale showing ridges and honeycomb lattice ending in trabeculae on top of a perforated multilayer lattice. Scale bars: (A) – 1cm, (B and C) – 1µm.Figure 2.Morphogenetic time series of the development of cuticular disks in pupal P. eurimedes dorsal forewing scales, acquired with a 100x confocal microscope. AF-555 WGA (green) stains chitin and Cellmask (red) stains plasma membrane. By 52% development, Hollow vein-like crossribs appear on plasma membrane in between ridges that serve as a scaffold for cuticle accretion. As scales mature, more cuticle is deposited into these rows of disks bounded by plasma membrane. See also Figs. S4- S6. (B-B’’’) Close up views of A-A’’’. (C-C’’’) xz cross-sections of the scale at locations marked with grey lines in A-A’’’ reveal the planar aspect of the cuticular disks. Yellow ROI in C’-C’’’ correspond to those in B’-B’’’. Scale bars (A-A’’’) – 5µm, (B-B’’’ and C- C’’’) – 2µm.Figure 3.Morphogenetic time series of the development of columnar honeycomb lattice in pupal P. eurimedes dorsal forewing scale cells acquired with super-resolution lattice SIM. AF-555 WGA (green) stains chitin and AF-647 phalloidin (red) stains F- actin. AF-555 WGA show a gradual evolution of the cuticular disks from filled-in planar to hollow tubular outgrowths. The disintegrating F-actin bundles show evidence of reorganization from linear to reticulated features with ring-like cross-sections. See also Figs. S7-S9. (B-B’’) Close up views of A-A’’. Insets correspond to the regions of interest (ROI) marked in yellow shown with a 3D aspect. (C-C’’) xz cross-sections of the scale at locations marked with grey lines in A-A’’. Scale bars (A-A’’) – 5µm, (B-B’’, C-C’’ and insets) – 1µm.Figure 4.Arp2/3 complex is involved in F-actin reorganization in pupal P. eurimedes wing scales. Dorsal forewing scales stained with AF-594 anti-Arp2 (green) and AF- 647 phalloidin (red) acquired with 100x confocal microscope. Initially (∼52% development), Arp2/3 complex appear as sparse punctate dots while the F-actin bundles are still intact. As F-actin bundles disintegrate and reorganize, a relatively higher density of punctate Arp2/3 signal is seen in close association with the reticulate F-actin network. Our oldest timepoint in P. eurimedes (∼76% development) has a large amount of cuticle autofluorescence overlapping with the AF594 signal, but the punctate pattern can be still discerned. See also Figs. S10-S12. (B-B’’) Close up views of A-A’’. (C-C’’) xz cross-sections of the scale at locations marked with grey lines in A-A’’. Yellow ROIs correspond to those in B-B’’. Scale bars (A-A’’) – 5µm, (B-B’’ and C-C’’) – 2µm.Figure 5.Conservation of honeycomb lattice development in genus Parides. SEM cross-section view of (A) adult green scales in P. arcas, (D) blue scales in P.nireus, and (G) green scales in P.palinurus. All pupal dorsal forewing scales are stained either with AF-555 WGA or FITC-WGA (green) showing chitin, and AF-647 phalloidin or TRITC-phalloidin (red) showing F-actin. (B-C) Maximum projected 3D-SIM micrograph of green cover scales in pupal male P. arcas featuring planar cuticular disks similar in shape and arrangement to P. eurimedes. (B’-C’) Maximum projected 3D-SIM micrograph of green cover scales of a different male P. arcas pupa showing irregular crossribs patterns. (E-F) Maximum projected 3D-SIM micrograph of blue cover scales of pupal P. nireus, similarly with crossribs and intact linear actin bundles. (H-I) 60x confocal micrographs of green cover scales of pupal P. palinurus shown at two different z-planes. A concave network of F-actin underlie the cuticular dimples. At lower z, the actin rings (in cross-section) are smaller in size and show a foam-like appearance. (C-C’, F, and I) xz cross-sections of the scales with ROI at locations marked with grey lines in B-B’, E, and H respectively. Scale bars (A, D, and G) – 5µm, (B-B’, C-C’, E, and F) – 1µm, (H-I) – 5µm.Figure 6.A schematic illustrating the proposed morphogenesis of honeycomb lattices in papilionid wing scales. During early stages, F-actin bundles prefigure the loci where ridges form in between adjacent actin bundles. Next the plasma membrane (black lines) forms a scaffold where cuticle (green lines and structures) accumulates into the spaces and forms irregular disk-like structures. Reorganization of F-actin around the disks subsequently extrudes the plasma membrane along with the deposited cuticle, forming the honeycomb lattice walls.