The Use of CRISPR-Cas9 Reveals the Major Genes that Control Wing Patterning and Coloration in Nymphalidae Butterflies

Abstract

Genes influence the colors and patterns on a butterfly’s wing. In particular, genes affect lamina thickness of wing scales, which controls the appearance of structural colors, and focal cells that determine the origin of certain patterns such as eyespots. This paper analyzes the spalt gene and genes from two major pigmentation pathways, the melanin biosynthesis pathway (yellow, ebony, tyrosine hydroxylase (TH), DOPA decarboxylase (DDC), and arylalkylamine N-acetyltransferase (aaNAT)) and the ommochrome synthesis pathway (vermilion, white, and scarlet), code for their roles in wing patterns, coloration, and morphology. Butterfly mutants of these genes generated using CRISPR-Cas9 are examined to understand the role of these genes in the overall appearance of the wing. The same gene deleted from several different species reveals if the function of that gene varies from species to species or if it remains the same. The feasibility of generating targeted genetic mutants using CRISPR technology has greatly advanced scientific studies of butterfly wing patterning and coloration. These butterfly knockout mutants display phenotypes that lead to the conclusions that spalt affects eyespot and chevron pattern development, and the melanin (but not ommochrome) biosynthesis genes affect scale melanization and morphology. These findings advance our understanding of the genetic regulation of butterfly wing patterning and coloration. This understanding can be used to propel future research that aims to artificially replicate these naturally occurring patterns and colors to create bio-mimetic materials.

Keywords: CRISPR; melanin pathway; eyespot; wing pattern; pigment; structural color; tyrosine hydroxylase; DOPA decarboxylase; aaNAT; spalt

Introduction

Within the animal kingdom, insects are the most diverse group in terms of their ample species numbers and various adaptations¹. These defining characteristics are shown in Lepidoptera, an order of winged insects that include butterflies, with their uniquely patterned and colorful scaled wings. Homologous pattern elements across Lepidoptera species include the border ocelli system, and marginal and submarginal bands, all of which run parallel to the wing edge². In Nymphalidae butterflies such as J. coenia, V. cardui, and B. anynana, the border ocelli system gives rise to eyespots. Although the colors of each wing on a butterfly may vary, the scales that make up these intricate wings comprise the same key components. Each scale has a gridded body plan, or Bauplan, with ridges running longitudinally along the length of the scale, which are joined by crossribs^3,4. That crossrib-ridge unit is supported by a structure called the lower lamina, which may vary in girth from scale to scale^3,4. Specific genes have been discovered to regulate the thickness of the lower lamina, which is known to influence (hue) coloration³.

The colors that appear on a butterfly’s wing derive from two types of colorants: pigments and structural colors. When organisms lack the pigments for certain hues, they use structural colors to achieve it. Pigments produce the traditional and natural coloring of animal tissues as the pigment molecules selectively absorb specific light wavelengths. In contrast, structural colors are produced when butterfly scale lamina, a photonic nanostructure, interacts with light³. These very tiny nanoscale structures can be seen using a specialized technique called Helium Ion Microscopy³. The lamina is composed of thin layers of chitin, and the total thickness of those layers altogether determine what structural color is produced^3,4. As light is reflected from both the topside and undersurface of the lamina, the reflections created interfere with each other^3,4,5. Depending on the lamina thickness and wavelength of light itself, the interference of these reflections may either be constructive, producing observable color, or destructive, creating a dull look to the resulting color³. In addition to producing a range of hues, structural colors also play roles in creating optical effects (such as creating iridescence in butterfly wings) and determining wing pattern elements (such as eyespots and marginal and submarginal color patterns) that together distinguish butterfly sexes, populations, and species³.

Pigments and structural colors were introduced as factors in color evolution as ways of evolving specific hues in times of diversification. The tuning of lamina thickness, in particular, was discovered to be a means of color evolution in both microevolution (minimal changes within a population over short time periods) and macroevolution (major changes of an entire classified group over long time periods). However, structural colors’ developmental and genetic origins are being actively investigated⁵.

Eyespots are prominent color pattern elements present on butterfly wings. Smaller eyespots located near the wing margin draw predators’ attention towards the edge of the wing and away from more vital areas in the case of an attack whilst larger eyespots act as an intimidation tactic in an effort to prevent attacks⁶. The circular ringed pattern of the eyespot is determined in the wing epidermis during the initial hours of pupal development, whereas the pigments are released into the scales only moments before adult emergence⁷. The cells located at the center (or focal point) of the eyespot are the focal cells, which serve as the source of positional information, essentially determining where the pattern develops^6,8. A morphogenic gradient with highest morphogen concentration at the focus is thought to be formed either by a gradient model or a sink model. Focal cells may secrete a morphogen,—a signaling factor responsible for morphological changes—and as that morphogen diffuses away from the center through gap-junctions, a radial concentration gradient is formed^6,7. Alternatively, a morphogenic gradient may be formed by focal cells acting like a sink for morphogen produced at high concentrations in the wing epidermis. Depending on the distance from the focus, cells are exposed to different morphogen concentrations during pattern determination, which affect the synthesis of specific pigments^6,7,7. For example, in the butterfly J. coenia, commonly known as the Buckeye, when morphogen concentration is at its maximum in the eyespot center, no pigment is produced, resulting in the white focal eyespot; when morphogen concentration is high but not yet at its peak, only black pigment is synthesized; when the concentration is low, a buff pigment—a brownish-yellow or tan color—is produced, resulting in the light colored ring; and at even lower levels, a brown pigment is produced, creating the eyespot’s brown peripheral ring⁶. This variation in pigment production affects the overall size and proportions of the eyespot⁷.

During early studies, scientists experimentally moved foci elsewhere on the wing to purposefully trigger the development of a pattern to occur in that chosen region, creating butterflies with new wing patterns, or variations of already existing ones⁸. Size and proportion of the eyespot feature had also been studied through artificial selection experiments⁹. However, the artificial selection method used in previous eyespot experiments was time consuming and expensive^7,10.

More recently, with the help of advancing technology, the genes suspected to play a role in the eyespot phenotypes have been further explored using the CRISPR-Cas9 gene editing system to understand their role in butterfly wing coloration/patterning. The CRISPR-Cas9 system has the ability to disrupt, delete, or insert a gene^11,12,¹³. CRISPR was first found as a part of an antiviral system in some bacterial species^11,12. In bacterial species, a guide RNA composed of two naturally occurring RNA molecules (spacer/repeat RNA and tracr RNA) marks the specific location where the Cas9 endonuclease acts as a pair of “molecular scissors”^11,12. Cas9 cuts just ahead of a short DNA sequence called protospacer-adjacent motif (PAM) site to break the bonds connecting the nucleotides in the DNA molecule^11,12,14. Scientists reengineered the CRISPR-Cas9 system as a gene-editing tool so that the CRISPR-Cas9 complex is “guided” to cut a desired target sequence by a synthetic gene editing molecule called single guide RNA (sgRNA)^11,12,14. The cell then attempts to repair the cut, using a process called non-homologous end joining, a process which often introduces mutations during repair of the cut ends. In the case of deleting a gene, two sgRNAs target and cut the DNA at separate sites. Non-homologous end joining fuses the two ends, creating a DNA sequence that lacks the targeted gene. Consequently, offspring and future generations that derive from the mutant organism will develop without expressing the deleted gene. Scientists applied this CRISPR-Cas9 mediated gene deletion strategy to butterflies, as this is a relatively precise gene editing method that allows scientists to modify site-specific areas and is significantly faster and cheaper compared to selective breeding procedures, calling the resulting mutants “mosaic butterflies” to account for the scattered colored regions on their wings that result from some cells having the target gene deleted and other cells not having the gene deleted¹⁴. The various phenotypes that resulted from knocking out genes that regulate wing patterning and coloration would then be analyzed, and hypotheses about those knockout genes’ functions/roles in wing color development would either be supported or opposed.

Wing patterns provide ecological advantages to butterflies, serving as camouflage for predator avoidance as well as identification for mating; therefore, understanding butterfly wing coloration is also vital to developing camouflage technologies for human use^2,15,16,17. Biological camouflage strategies have inspired the creation of several camouflage devices used in the military today^18,19. For example, the light absorption nanostructure in butterfly wing scales inspired the development of porous anodic aluminum flake powder, which mimics the low reflectance visual camouflage demonstrated in butterflies, and since the structure is dependent on light, the camouflage can operate omnidirectionally²⁰. The development of these bio-mimetic materials that achieve the natural properties of a butterfly wing—light absorption, structural color display, reflection—are not confined to the militaristic lifestyle; they also extend into ordinary, everyday lives²¹. Some butterfly wings are noticeably bright and colorful even when subjected to direct sunlight. This crisp illumination has inspired scientists to strive to mimic this effect in electronic screens by creating screens that resist the washed out colors created when sunlight makes contact with the screen^22,23.

In this paper, genes suspected to play a role in wing development are examined using CRISPR-Cas9 generated mutants. Specifically, the spalt gene, one of the earliest genes expressed in eyespot focal cells, and genes from the major pigmentation pathways–melanin and ommochrome pathways–are examined. The research discussed in this paper addresses the question: do the spalt gene and genes from the melanin biosynthesis pathway–yellow, ebony, tyrosine hydroxylase (TH), DOPA decarboxylase (DDC), arylalkylamine N-acetyltransferase (aaNAT)–and the ommochrome synthesis pathway–vermilion, white, and scarlet–influence the presence or absence of wing patterns, coloration, and morphology?

Results

Spalt Gene Affects Eyespot Pattern

The spalt gene is a transcription factor expressed during the last developmental instar stage in the region where the focus of the eyespot will presumably form in B. anynana and J. coenia². It is one of the earliest genes expressed in eyespot focal cells across many butterfly species, which suggests that it could be a critical regulator of eyespot patterning. Recent studies using CRISPR-Cas9 have provided greater understanding of spalt function in eyespot patterning.

Deletion in the spalt gene using CRISPR-Cas9 resulted in a reduction or complete loss of color patterns of the eyespot, producing atypical wing phenotypes in mutant individuals²⁴. In many J. coenia mutants, eyespots were completely missing from the forewing (Figure 1a, b), hindwing (Figure 1c), or both regions, suggesting that spalt is an important factor in eyespot determination in this species²⁴. Among the 21 J. coenia adult mutants generated, there were 16 total reduced eyespots and 20 total lost eyespots²⁴. Eyespots that develop in the same position but located on opposing wing surfaces are determined separately, meaning that a missing eyespot on the ventral surface does not necessarily mean that the eyespot will be missing on the dorsal side and vice versa (Figure 1b)²⁴. As seen in Figure 1d, Vanessa cardui mutants also showed reduced eyespots (missing rings and pigment in the eyespot appearance, but there is still a trace of a pattern). Among the 14 V. cardui adult mutants generated, 11 eyespots were reduced, with only 1 mutant showing eyespot loss (a completely blank area with no traces of a pattern present)²⁴. Since these are mosaic knockouts, each mutant examined has the gene removed from different cells, which may account for the varying phenotypes across the different species²⁴. In all instances across both species, only the eyespot patterns were affected, whereas adjacent color patterns retained their characteristics, indicating spalt regulates the eyespot pattern. The eyespots in both species often appeared split down the middle due to the presence of abnormal wing veins (Figure 1e, f), a mutant phenotype consistent with that seen in the fruit fly Drosphilia melanogaster mutants lacking spalt^24,25. Vein abnormalities were also found in areas around the discal cell along with decreased discal spot patterns in J. coenia mutants. However, these defects were thought to be unrelated to the effects of spalt deletion as the gene does not express in discal spot patterns^24,26. The missing and reduced eyespot phenotypes exhibited by spalt deletion mutants in J. coenia and V. cardui indicate that spalt positively regulates eyespots.

Spalt Affects Other Color Patterns and Scale Melanization

The wing margin in an adult butterfly is a region on a butterfly’s wing with a strong dependency on spalt². During the last developmental instar stage, spalt is expressed in the wing disc cells located along the border lacuna that later emerge as the margin on an adult wing². When the gene was deleted in J. coenia, the “W” shaped submarginal band color patterns were either disrupted or lost in the hindwings and forewings (Figure 2a-e). In particular, the EIII submarginal band was disturbed in spalt mutants, while the neighboring EI and EII submarginal bands appeared unaffected. This disruption was also associated with the reduction/loss of eyespots seen in many mutants that displayed both phenotypes².

Melanin Biosynthesis Pathway Genes’ Role in Color Regulation

CRISPR-Cas9 knockout experiments on a variety of butterfly species revealed the genes responsible for regulating color and morphology in butterfly wing scales and their slightly different effects in each species. By focusing on enzymes from the melanin pathway and the ommochrome synthesis pathway, the specific enzymes and products required for creating each of the colors seen in butterfly wing scales were revealed. The wings of B. anynana butterflies consist of gold, beige, brown, and black pigments that are thought to result from this pigmentation pathway as well as the ommochrome pathway discussed later in this paper¹⁷. J. coenia butterflies are mostly brown with an orange band and a brown and white margin; the eyespots consist of black and white outer rings and a multicolored center, including black, blue, orange, and magenta colors. V. cardui butterflies have an orange–brown background color with white spots on the forewing and rows of black spots on the hindwing. The melanin pathway can produce five different molecules, including two types of eumelanin (dopa-melanin, which produces black pigments, and dopamine-melanin, which produces brown pigments), pheomelanin (a reddish-yellow pigment), N-?-alanyl dopamine (yellow), and N-acetyl dopamine (colorless) (Figure 3A)¹⁷. Five genes from the melanin pathway were targeted for deletion using the CRISPR-Cas9 system: yellow, ebony, tyrosine hydroxylase (TH), DOPA decarboxylase (DDC), arylalkylamine N-acetyltransferase (aaNAT). By analyzing these mutants, an understanding of the subset of melanin pathway genes needed to produce each of the colored scales was deduced (Figure 4A). For example, scales expressing TH, yellow, and DDC make dopa melanin and dopamine-melanin, which result in a brown or black colored scale; scales expressing TH, DDC, and aaNAT make NADA sclerotin, which results in a white colored scale; scales expressing TH, DDC, and ebony make dopamine-melanin and NBAD sclerotin, which result in a beige colored scale; and scales expressing TH, DDC, and ebony may also make pheomelanin alongside dopamine-melanin and NBAD sclerotin, which result in a gold colored scale. To ensure the observed phenotypes are due to direct, cell-autonomous effects in wing scale development rather than adverse effects of disturbing the whole organism, prior analyses on the wing scales and organisms were conducted; post-knockout, the mutants were noted to develop normally except the wings, which were altered¹⁷.

TH Mutants

The first step of the melanin pathway involves TH catalyzing tyrosine into DOPA¹⁷. Consequently, knocking out the TH enzyme and disrupting its intended function eliminates all the melanin pathway products in B. anynana as well as several other arthropods. Three out of the 21 experimental larvae had a lack of black pigment on their normally black head capsule, indicating that TH is necessary for larval head pigmentation¹⁷. The TH mutants displayed missing colors on their wing scales, other clones’ scales were less pigmented and curled, and white scales were absent altogether, meaning that the TH gene is also required for the overall pigmentation, development, and structural rigidity of scales¹⁷. In J. coenia, a deficit of TH resulted in amelanism in emerged scales, the effects varying depending on the region: scales, originally dark brown, on the thorax became white; most wing mutant scales became lighter in color; and melanic scales of the inner rings of the eyespot turned completely transparent. The foci of J. coenia are characteristically blue, but mutant eyespots did not develop this feature because the black ground scales—a class of scales also known as pigmentary scales that form a “ground” pattern and color layer on a butterfly’s wing²—became transparent, and the cover scales responsible for creating the blue hue lack the black background required for blue reflectance/iridescence²⁷.

DDC Mutants

The function of DDC is to catalyze the conversion of DOPA into dopamine¹⁷. Disrupting this function resulted in a lack of black pigment in five of the 42 larvae. A total of seven butterflies emerged, two of which displayed color disruptions on their eyespot scales: gray scales in place of the originally black ones, whitish scales rather than brown and beige ones, a paler gold ring, and a total lack of white center scales. This absence suggests that dopamine is a necessary molecule for white scale development. Compared to the wild-type scales and disregarding the beige scales, DDC mutant wing scales were substantially lighter and less yellow, indicating that dopamine is an essential factor for the pigments in wing scales¹⁷. Although lighter in tone, black and gold pigments were still present in the mutants’ scales, meaning that dopa-melanin and non-dopamine-derived pigments were also present in the black and gold scales, respectively. Like the TH mutants, DDC mutated scales had a curled appearance, indicating that DDC is also required for scale rigidity^17,28. These color changes were supported by L*a*b color space analyses, a means of representing color consisting of three axes: L, representing darkness to lightness; a, representing greenness to redness; and b, representing blueness to yellowness¹⁷. Only two V. cardui mutant butterflies were generated that also displayed heavy depigmentation in black scales and unaffected red scales. A large number of the larvae presented decreased melanin phenotypes, reinforcing DDC’s importance in melanin synthesis²⁷.

aaNAT Mutants

aaNAT catalyzes the production of colorless N-acetyl dopamine (NADA) sclerotin from dopamine¹⁷. The head capsules of wild type B. anynana are typically black in the first and second instars but naturally change to a light brown color from the third instar into the fourth. This normal black colored head capsule was shown in 20 larvae in their third instar, but during their last larval instar, 16 retained the black color, meaning that aaNAT is required for brown pigmentation in the head capsules of late larvae¹⁷. Multiple eyespot centers also lacked white scales in four out of the 20 butterflies, indicating that aaNAT is necessary for the development of those central scales²⁸.

yellow and ebony Mutants

The originally black and brown scales in wild-type B. anynana butterflies were significantly lighter in the yellow mutants, while the gold, beige, and white scales showed no change in color, demonstrating that yellow is only active in the darker colored scales as it converts DOPA to dopa-melanin^17,28. Similarly, yellow mutants of V. cardui showed a strong decrease in melanins. A lack of pigment in larvae head capsules was also observed in both V. cardui and B. anynana butterflies²⁷. Red colored scales were not greatly affected by yellow mutations, meaning the gene has little or zero effect on the deposition of non melanin derived pigments, and it plays a primary role in synthesizing black melanin pigments²⁷. Ebony mutants of B. anynana displayed results contrary to that of the yellow mutants with light colored scales on wild-types being significantly darker on the mutants and dark scales having no change in color; this suggests that ebony is only active in the light colored scales as it converts dopa-melanin to yellow N-?-alanyl dopamine (NBAD) sclerotin¹⁷. Hence, removing ebony makes these gold and beige scales darker and less yellow in color in B. anynana^17,28. Similarly, ebony deletion in V. cardui mutant adults’ wings also showed darkened wing phenotypes. In V. cardui pupa wings of ebony mutants, the red central band, white eyespot region, and brown marginal band turned black; the adults, however, did not display any of these phenotypes. J. coenia mutant adults’ wings also showed clusters of dark scales, but the overall dorsal brown background color was not affected²⁷.In B. anynana, J. coenia, and V. cardui, red–orange scales and buff/yellow scales were consistently affected by the deletion of ebony²⁷. The varying effects of yellow and ebony between species may indicate that factors other than those of the melanin biosynthesis pathway regulate these subtle differences in scale structure and color.

Melanin Biosynthesis Pathway Genes Affect Scale Morphology

Deleting the melanin pathway genes affected not only coloration of scales but also scale morphology²⁸. In yellow mutants of B. anynana, a supernumerary lamina covered the windows of the brown and black scales, a rare occurrence in wild-type scales and an abnormal phenomenon overall, with the exception of the white scales, which normally display this lamina¹⁷. The white, beige, and gold scales of the yellow mutants displayed no abnormalities. The crossrib structure in these mutant scales were thinner and the spacing notably denser in the darker colored scales, but the distance between the longitudinal ridges and the scale size as a whole were unaltered¹⁷. DDC mutants of B. anynana had disordered crossrib arrangement, thinner crossribs, and often fused crossribs, which resulted in bigger windows (Figure 6)¹⁷. The trabeculae, a supporting tissue element located beneath the crossribs of a butterfly scale, was taller in the mutants relative to that of the wild-type. The overall scale size and distance between ridges, however, remained the same as seen in the yellow mutants. A subtle increase in scale size of black and beige scales were observed in the ebony mutants of B. anynana along with slightly thicker crossribs in the brown scales and a greater distance between ridges in white and gold scales. The observed morphological changes in ebony mutants were rather minimal—having a negligible effect on size, color, and morphology transformation from one colored scale to another—and may simply be a result of the natural genetic variety between the measured individuals¹⁷. The wing scales of J. coenia pale mutants (pale is encoded by TH) became rounded in place of the natural scalloping texture and melanic scales of the inner rings of the eyespots underwent a thickness reduction or structural defect²⁷.

Concisely, the darker scales (brown and black) of yellow mutants and all colored scales of DDC mutants showed drastic changes in color and morphology compared to those of the wild type butterflies. The color changes in ebony mutants were notable, but its scale morphology changes were rather small in comparison. J. coenia butterflies had some rounded scales and some regions appear disheveled with thinner scales due to structural flaws.

Ommochrome Biosynthesis Pathway Genes

The ommochrome pathway, another major pigment pathway found in butterflies, produces red, orange, and yellow pigments (Figure 3B)¹⁷. A previous study on B. anynana showed vermillion expression in the wing during development, which suggested that vermillion and the ommochrome pathway could function in wing pattern and/or color²⁹. Three genes from the ommochrome pathway–vermillion, white, scarlet–were investigated for their roles in butterfly wing development using CRISPR-Cas9 technology.

All three genes from the ommochrome biosynthesis pathway were concluded irrelevant in determining wing color and pattern in B. anynana as their mutants did not display any color or morphological disruptions in their wings, and the ommochrome pathway is thought to have no contribution to the gold color in B. anynana butterflies; ommochromes are still present in the wings despite the enzymes’ functions being absent^17,27,30.

The colored scales of V. cardui wings were also unaffected by white and scarlet³⁰. However, color disruption was observed in the body color of 12 of the 58 hatched white larvae mutants along with a lack of pigment in the eyes of adults, and the eye of a single adult scarlet mutant also displayed this lack of pigmentation¹⁷. During pupal development, vermilion is expressed in the pigment cells of an ommatidia—the unit that composes an arthropod’s compound eyes—, synthesizing pigment; experiments that knocked out this gene in B. anynana created mutant adults with yellow or pink eyes compared to the black eyes of the wild-type butterflies³⁰.

Discussion

In this paper, the intricate wing patterning and coloration of butterflies is reviewed. The notable role that CRISPR technology played in understanding the role of genes responsible for this phenomenon is acknowledged^{2,31,17,24,27,28,30}. We describe results obtained by generating CRISPR knockout mosaic mutants of genes previously suspected to play a role in wing development. In brief, the spalt gene and genes in the melanin biosynthesis pathway, TH, DDC, aaNAT, yellow, and ebony have been found to be important for wing pattern development whilst those from the ommochrome pathway, vermilion, scarlet, and white, have been shown to have no effect on wing development¹⁷. However, the ommochrome pathway genes were found to affect eye pigmentation³⁰.

With the invention of CRISPR-Cas9 gene editing system, scientists have identified spalt as an important contributor to the development of eyespots, as well as marginal and submarginal patterning, and scale melanization in several butterfly species²⁴. All butterfly eyespots are formed by a shared developmental mechanism, but the findings regarding this single gene show that the diversity of eyespots across species evolved through genetic changes within this mechanism^7,8,9,32,33. Several minor eyespot patterning genes create the slightly phenotypically different eyespots; these differences are indicators of divergence amongst the most closely related species³².

These studies only studied spalt gene deletions in a few species of Nymphalidae butterflies. Future studies could look at additional species in this family for corroborating evidence as well as examine the gene in other butterfly families to explore if spalt function is conserved across butterfly families or just within species. The main focus of these spalt studies is the gene’s relation to determination of an eyespot. However, experiments also resulted in disrupted submarginal color patterns. Later studies could further investigate and focus on spalt function in butterfly wing margins. Vein abnormalities also arose in spalt J. coenia mutants and could be examined further in the same species as well as others to determine the relation, if any, between spalt and vein development.

Genes in the melanin biosynthesis pathway, TH, DDC, aaNAT, yellow, and ebony, were found to be important for wing pattern development. Expression of particular subsets of melanin pathway genes in a cell was deduced to give rise to the resulting color in that cell. Across all the melanin pathway knockout experiments, these mutants showed amelanism, missing white scales, lack of brown pigmentation, and darker colored scales. In addition to its function in pigmentation, DDC and yellow were also shown to affect scale morphology. The loss of dopamine-melanin and dopa-melanin when DDC and yellow were knocked out in the butterfly wing is though to impede the polymerization of cuticular protein around the crossribs, causing these scale morphology changes. In insects, melanin pathway products are known to play a role in hardening the cuticle that forms the insect exoskeleton. As such, it is suspected that DDC and yellow may play a similar role in hardening crossrib structures in the butterfly wing. Moreover, deposition of melanin pigments coincides with the development of crossribs and other fine structures during late pupal development.

Surprisingly, genes from the ommochrome pathway, vermilion, scarlet, and white, were shown to have no effect on wing development despite past studies indicating their expression in the developing wing¹⁷. This may be because CRISPR did not knock out the ommochrome pathway genes in the wing cells of the butterfly knockout mutants but removed ommochrome genes from other cells in the insect, such as eye cells (which may be why irregular eye phenotypes were expressed)¹⁷. The ommochrome pathway may also not be the only pathway that determines particular wing phenotypes, meaning that knocking out the ommochrome genes does not halt wing pattern development because another redundant pathway contains genes that also produce the same phenotypes. However, the ommochrome pathway genes were found to play an important role in eye development, in which gene deletion was shown to affect eye pigmentation in these mutants³⁰.

This paper solely discusses single, mosaic knockouts as a literature search using keywords “yellow gene butterfly double knockout”, “TH gene butterfly double knockout”, “DDC gene butterfly double knockout” (along with separate searches replacing “double” with “rescue”) and “melanin and butterfly” and “melanin and wing” did not indicate that double nor rescue knockout experiments on these select genes in these select species have been done; however, performing double and rescue knockout experiments for future research would clarify the necessity and sufficiency of these select genes. The biosynthesis pathways studies mentioned in this paper only researched genes in butterflies with simpler morphologies. Other studies could look at these genes in other butterflies with iridescent structural colors and more complex morphologies; further investigations could determine if those genes contribute to the creation of complex photonic materials in butterfly wing scales¹⁷. Future studies could also examine the melanin pathway genes in other butterfly families as well as other species within the Nymphalidae family to establish common or varying functions across families and species. Studies focusing on the ommochrome pathway could examine other genes since vermilion, white, and scarlet were deemed unnecessary to wing pattern determination and color production.

Studying the development of wing patterning and coloration contributes to a deeper understanding of the evolutionary processes of Lepidopteras and maintenance of a healthy adaptive ecosystem. Butterflies are beneficial to the environment, acting as pollinators for plants³⁴ and indicators of a healthy ecosystem³⁴, and increasing their longevity only ensures the health of the planet. Artificially engineered wing patterns may provide butterflies with a more advantageous form of camouflage against their predators. Furthermore, the use of these findings may help create biomimetic technologies advantageous to citizens and military personnel. Future studies and discoveries regarding wing patterning and the genes responsible for it will propel humanity onto a whole new level of bioengineering.

Conclusion

This paper focuses on the use of CRISPR-Cas9 technology in butterfly knockout experiments and summarizes the findings of a few select genes–spalt, yellow, ebony, TH, DDC, aaNAT, vermilion, white, scarlet–known to influence butterfly wing coloration and patterning. By deleting a chosen gene from individuals, scientists compare the mutant to the wild-type and are able to narrow down, if not determine, the function of the gene. The deletion of spalt in wing scales of J. coenia and V. cardui resulted in total loss of or reduced eyespots, respectively, leading to the conclusion that spalt does influence the presence of eyespots on butterfly wings. The genes from the melanin biosynthesis pathways were found to affect melanin production in wing scales: deletion of TH resulted in amelanism in head capsules of B. anynana mutants and wing and ground scales of  J. coenia mutants, deletion of DDC resulted in amelanism in eyespot scales, deletion of aaNAT resulted in no brown pigment production in larvae head capsules, deletion of yellow resulted in lighter black and brown scales, deletion of ebony resulted in darker gold and beige scales. Melanin pathway genes were also concluded to influence scale morphology in B. anynana mutants: TH and DDC mutants produced curled scales, yellow mutants developed a supernumerary lamina and crossrib structure was thinner, and DDC mutants had disordered crossrib arrangement. These results indicate that these melanin pathway genes influence wing coloration and morphology. The ommochrome pathway genes vermilion, white, and scarlet do not influence scale morphology and wing color and pattern determination as the mutants still contained ommochromes. These discoveries solidify previous research and present new findings. The continuous study of butterfly wing pattern and color development contributes to a deeper understanding of the evolutionary processes of Lepidopteras and inspire engineers to produce biomimetic technologies.

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