Robert D. Reed

Research on butterflies has uncovered a conserved “toolkit” of genes for color pattern development and evolution. One of these genes is optix , a homeobox transcription factor that regulates ommochrome and melanin pigmentation, as well as structural coloration, in nymphalid butterflies. It remains unclear, however, whether optix plays any roles in color patterning outside of the Nymphalidae. We used CRISPR‐Cas9 to disrupt optix in the tobacco hornwormmoth Manduca sexta and observed a dramatic abdominal pigmentation phenotype, where orange pigmentation was replaced by black eumelanin. Chemical assays suggest that the orange pigment is not an ommochrome, indicating that optix modulates an alternative, uncharacterized pigment pathway in M. sexta . RNA‐seq and chemical analyses of orange and black abdominal scales lead us to speculate that the orange pigment may be a type of melanin, perhaps N‐β‐alanyldopamine (NBAD) sclerotin. Our results suggest that optix plays a deeply ancestral role in pigment regulation in Lepidoptera, and demonstrates evolutionary flexibility in how it interfaces with pigment chemistry across moths and butterflies.

Research on butterflies has uncovered a conserved "toolkit" of genes for color pattern development and evolution. One of these genes is optix , a homeobox transcription factor that regulates ommochrome and melanin pigmentation, as well as structural coloration, in nymphalid butterflies. It remains unclear, however, whether optix plays any roles in color patterning outside of the Nymphalidae. We used CRISPR-Cas9 to disrupt optix in the tobacco hornwormmoth Manduca sexta and observed a dramatic abdominal pigmentation phenotype, where orange pigmentation was replaced by black eumelanin. Chemical assays suggest that the orange pigment is not an ommochrome, indicating that optix modulates an alternative, uncharacterized pigment pathway in M. sexta . RNA-seq and chemical analyses of orange and black abdominal scales lead us to speculate that the orange pigment may be a type of melanin, perhaps N-β-alanyldopamine (NBAD) sclerotin. Our results suggest that optix plays a deeply ancestral role in pigment regulation in Lepidoptera, and demonstrates evolutionary flexibility in how it interfaces with pigment chemistry across moths and butterflies.

Butterfly wings present a tremendous gallery of colorful patterns, offering a unique opportunity to study how developmental pattern formation processes evolve. We still do not understand the genetic basis of several key aspects of wing pattern development, however. Three paralogous POU domain transcription factors nubbin, ventral veinless (vvl), and pdm3 are all known wing development genes in Drosophila melanogaster. Here we combine gene expression and knockout approaches to show that each of these genes plays multiple novel wing patterning roles in the common buckeye butterfly, Junonia coenia. We found that nubbin controls eyespot pattern determination via a non-cell autonomous repressor-like effect originating at the wing veins, such that nubbin knockouts have larger eyespots. nubbin also regulates pigment identity and scale morphology across the wing. We also found that vvl regulates pigment identity of the discal bands and ventral hindwing. Last, we found that pdm3 is required for determining the outer rings of eyespot patterns, where it is co-expressed with spalt and the lncRNA ivory. pdm3 is also necessary for determining wing margin stripes, where it is again co-expressed with spalt, leading us to propose that the eyespot and wing margin gene regulatory networks could be homologous. Finally, pdm3 affects pigmentation of the ventral hindwing, phenocopying the seasonally-plastic color switch in J. coenia. Together, our work shows that POU domain transcription factors play diverse roles in butterfly wing pattern development and highlights nubbin as one of the first genes implicated in the repressive function of wing veins in color pattern determination.

Butterfly wings exhibit a remarkable diversity of structural iridescent colours, yet the genetic regulation of iridescence remains poorly understood. Here, we show that the Iroquois-complex transcription factor gene araucan plays a role in modulating wing scale iridescence in the common buckeye butterfly, Junonia coenia. Using CRISPR-Cas9 knockouts, we demonstrate that loss of araucan function causes dorsal wing scales to shift from golden-brown to blue iridescence, and eyespot centre scales to shift from saturated purple-violet to dull grey-brown. These phenotypes are associated with increased lower lamina thickness (LL) in dorsal cover scales and decreased LL in eyespot-centre cover and ground scales, effects known to influence thin-film interference iridescence, along with reduced pigmentation in eyespot ground scales. Together, these results identify araucan as a single transcription factor that determines colouration by simultaneously regulating both photonic architecture and light-absorbing pigmentation and show that these effects can be regulated in a pattern-specific manner. Our findings provide a framework for linking gene regulation to the spatially coordinated evolution of structural and pigmentary components of colour.

Insect wings, a key innovation that contributed to the explosive diversification of insects, are recognized for their remarkable variation and many splendid adaptations. Classical morphological work subdivides insect wings into several distinct domains along the anteroposterior (AP) axis, each of which can evolve relatively independently to produce the myriad forms we see in nature. Important insights into AP subdivision of insect wings come from work in Drosophila melanogaster; however, they do not fully explain the diversity of AP domains observed across broad-winged insects. Here, we show that the transcription factor mirror acts as a selector gene to differentiate a far posterior domain in the butterfly wing, classically defined as the vannus, and has effects on wing shape, scale morphology, and color pattern. Our results support models of how selector genes may facilitate evolutionarily individuation of distinct AP domains in insect wings outside of Drosophila and suggest that the D. melanogaster wing blade has been reduced to represent only a portion of the archetypal insect wing.

Insect wings, a key innovation that contributed to the explosive diversification of insects, are recognized for their remarkable variation and many splendid adaptations. Classical morphological work subdivides insect wings into several distinct domains along the antero-posterior (AP) axis, each of which can evolve relatively independently to produce the myriad forms we see in nature. Important insights into AP subdivision of insect wings comes from work in Drosophila melanogaster, however they do not fully explain the diversity of AP domains observed across broad winged insects. Here we show that the transcription factor mirror acts as a selector gene to differentiate a far posterior domain in the butterfly wing, classically defined as the vannus, and has effects on wing shape, scale morphology, and color pattern. Our results support models of how selector genes may facilitate evolutionarily individuation of distinct AP domains in insect wings outside of Drosophila, and suggest that the D. melanogaster wing blade has been reduced to represent only a portion of the archetypal insect wing.

The evolution of sexual secondary characteristics necessitates regulatory factors that confer sexual identity to differentiating tissues and cells. In Colias eurytheme butterflies, males exhibit two specialized wing scale types — UV-iridescent (UVI) and lanceolate scales — absent in females and likely integral to male courtship behavior. This study investigates the regulatory mechanisms and single-nucleus transcriptomics underlying these two sexually dimorphic cell types during wing development. We show that Doublesex (Dsx) expression is itself dimorphic and required to repress the UVI cell state in females, while unexpectedly, UVI activation in males is independent from Dsx. In the melanic marginal band, Dsx is required in each sex to enforce the presence of lanceolate scales in males, and their absence in females. Single-nucleus RNAseq reveals that UV-iridescent and lanceolate scale cell precursors each show distinctive gene expression profiles at 40% of pupal development, with marker genes that include regulators of transcription, cell signaling, cytoskeletal patterning, and chitin secretion. Both male-specific cell types share a low expression of the Bric-a-brac ( Bab ) transcription factor, a key repressor of the UVI fate. Bab ChIP-seq profiling suggests that Bab binds the cis-regulatory regions of gene markers associated to UVI fate, including potential effector genes involved in the regulation of cytoskeletal processes and chitin secretion, and loci showing signatures of recent selective sweeps in an UVI-polymorphic population. These findings open new avenues for exploring wing patterning and scale development, shedding light on the mechanisms driving the specification of sex-specific cell states and the differentiation of specialized cell ultrastructures.

Long noncoding RNAs (lncRNAs) are transcribed elements increasingly recognized for their roles in regulating gene expression. Thus far, however, we have little understanding of how lncRNAs contribute to evolution and adaptation. Here, we show that a conserved lncRNA, ivory , is an important color patterning gene in the buckeye butterfly Junonia coenia . ivory overlaps with cortex , a locus linked to multiple cases of crypsis and mimicry in Lepidoptera. Along with a companion paper by Livraghi et al., we argue that ivory , not cortex , is the color pattern gene of interest at this locus. In J. coenia , a cluster of cis -regulatory elements (CREs) in the first intron of ivory are genetically associated with natural variation in seasonal color pattern plasticity, and targeted deletions of these CREs phenocopy seasonal phenotypes. Deletions of different ivory CREs produce other distinct phenotypes as well, including loss of melanic eyespot rings, and positive and negative changes in overall wing pigmentation. We show that the color pattern transcription factors Spineless, Bric-a-brac, and Ftz-f1 bind to the ivory promoter during wing pattern development, suggesting that they directly regulate ivory . This case study demonstrates how cis -regulation of a single noncoding RNA can exert diverse and nuanced effects on the evolution and development of color patterns, including modulating seasonally plastic color patterns.

Evolutionary variation in the wing pigmentation of butterflies and moths offers striking examples of adaptation by crypsis and mimicry. The cortex locus has been independently mapped as the locus controlling color polymorphisms in 15 lepidopteran species, suggesting that it acts as a genomic hotspot for the diversification of wing patterns, but functional validation through protein-coding knockouts has proven difficult to obtain. Our study unveils the role of a long noncoding RNA (lncRNA) which we name ivory , transcribed from the cortex locus, in modulating color patterning in butterflies. Strikingly, ivory expression prefigures most melanic patterns during pupal development, suggesting an early developmental role in specifying scale identity. To test this, we generated CRISPR mosaic knock-outs in five nymphalid butterfly species and show that ivory mutagenesis yields transformations of dark pigmented scales into white or light-colored scales. Genotyping of Vanessa cardui germline mutants associates these phenotypes to small on-target deletions at the conserved first exon of ivory . In contrast, cortex germline mutant butterflies with confirmed null alleles lack any wing phenotype and exclude a color patterning role for this adjacent gene. Overall, these results show that a lncRNA gene acts as a master switch of color pattern specification and played key roles in the adaptive diversification of wing patterns in butterflies.