Fig. 1. optix determines wing scale color identity and morphology in H. erato and A. vanilla. (A) optix mosaic knockouts in Marato result in con- version of red ommochrome color patterns to black melanin. The compari sons shown are left-right asymmetrical knockout effects from single Individual injected butterflies() Detail of mutant clone highlighted in the mutant in A showing red replaced by black in a proximal red "dennis" pattern of the dorsal forewing (C-C) optix knockout mosaics showing transformation of pointed wing conjugation scales to normal wing scales. Each panel in the series shows successive detail. (D) optix replaces orange and brown ommochromes in A. vanilla with melanin, resulting in a black and silver butterfly. Arrows highlight presumptive clone boundaries dis- cussed in the text. ) Detail of a knockout clone boundary highlighting the witch between red and black pigmentation in the ventral forewing from D. F) Ventral view of black spots in optix knockout mutant showing a phe- notype similar to WT. (G and G) Wing conjugation scales in WT (G) and optix knockout mutant (G) demonstrating a role for opt in determining A. vanile scale morphology y black pigments. These results show that optiris re- replaced by quired for red color pattern specification in Heat, and acts as a coordinating "or" switch between ommochrome (orange and pigment types red) and melanin (black and gin color patterning in a more To to test whether optic has a role basal heliconiine butterfly, we generated knockouts in the gulf fritillary A. vanille (Fig. 1 D-F, Fig. S1, and Dataset $1, Tables S1 and S2). Previous in situ hybridization work in 4 vanillae suggested that optix is not expressed in association with ommo- chrome patterns during early pupal development, leading to the hypothesis that the gene might not play a major color patterning role in this species (14). Thus, we were surprised to find that optix knockout resulted in a a complete transformation of Commo chrome scales to black melamin scales, producing a very unusual and dramatic phenotype of a completely black and silver butterfly (Fig. ID). We also observed a handful of oran orange or scales that changed to silver patches (Fig. 1D, ventral forening, green arrows) although we cannot confidently conclude that these autonomous knockout effects since it has been shown that silver scales can be induced through long-range signaling (18, 19), in this case potentially from neighboring knockout dones. The wild-type (WT) black spots and marginal bands in the ventral forewing were unaffected in knockouts and remained a darker color relative to the neighboring mutant melanic scales (Fig. 1F), optér knockout also resulted in melanie hyperpigmentation in adult bodies (Fig. S14). Thus, our results in a vanillae are consistent with those in Herato in supporting a role for optir as a switch-like regulator that toggles between ommochrome and melanin patterns or brown: in We next aimed to test whether optic regulates wing patterning malines V. candi (Fig. 2, Fig 52, and Duaset SL, Tables and Dataset SI, Tables si and S2) and J. conia (Fig. 3, Fig. S3, and Dataset S1, Tables S1 and S2), which diverged from heliconiines by-75-80 mya (20, 21). Our results were consistent with those from H. ento and A vanillae, where opár knockouts in both species showed mutant clones with complete loss of presumptive ommochrome pig ments and replacement by melanins (Figs. 2A-E and 3 A-C) One interesting exception to this finding was in 1 card, where the complete ommochrome-to-melanin switch consistently oc- curred in dorsal wings (Fig. 2.4 and B), but much of the ventral wing area showed only a loss of ommochrome and little obvious hypermelanization (Fig. 2A, C, and D). Importantly, however, we recovered late-stage papal wings from V. cand that had died before concigence that displayed hypermclamation of ventral wing surfaces (Fig. 3E). We speculate that this variable strength of ventral wing pattern melanization among individuals may re- flect a dosage effect, with the the stronger phenotypes representing biallelic optix deletion clones. We have no direct evidence for this, however, given the challenges in rigorously characterizing specific alleles from individual mutant clones (16). We also re- covered hypermeanic optic knockout in both V. candu pupae in (Fig S2) and J. conia (Fig. S3) 53). Taken together, our our knockout data from four nymphalids clearly demonstrate that optix plays a conserved role in coordinating the color identities of butterfly wing scales, where it operates as an "or" function between ommochrome and melanin identities, but also may be modulated to serve as an "and" function in some contexts, as demonstrated by phenotypes seen in the ventral wings of V. condui optix Function Is Required for Determination of Derived Scale Structures Along with its expression in color patterns, in situ optix expression also precisely predicts the location of patches of derived, pointed scales thought to play a role in conjugating forewings and hindwings during fight (5, 14). To determine whether optix plays a role in the unusual morphology of these scales, we examined op knockouts for in wing scale structure. Indeed, we d found that in all four species, op knockout resulted in trans formation of wing conjugation scales to normal wing scales (Figs. 1 C and G, 2F, and 3F). Furthermore, in H. enato, A. vanillae, and V. candi, where wing conjugation scales display color pigmentation we observed both structural and pigmentation changes in the same scales, suggesting that optic can can coregulate both scale scale morphology and pigmentation simultaneously. One final observation of note relates to the op-expressing pheroscales that occur along the veins of male Avanie (14). These scales did not show any grossly a parent transformation in optir knockouts (data not shown), even though the scales occurred within obvious knockout dones Therefore, whether optic plays a functional role in the development of pheroscales, as was predicted previously (14), remains an open question. In sum, our observations that optr knockout results in transformation of wing conjugation scales to normal wing scales ap Fig. 2. optix determines wing scale color identity and morphology in card (A) optix knockout mutant showing loss of ormochrome pigments (8-0) Left-right ammetrical comparisons from individual optix mutant but terflies, showing melanization of red patterns, loss of color pigmentation without widespread hyperelanization in the ventral forewing (C) and hindwing (D)) Severe defects in late-stage pupal wings displaying hypermelanization in red regions of donal and ventral wing surfaces (green and purple arrowhead compared with mosaic adult mutants in A. (F) optix knockout showing con- version of pointed wing conjugation scales to normal scales