Color pattern formation is a key model for studying evolutionary and developmental mechanisms. In the fruit fly Drosophila guttifera, which exhibits distinctive polka-dot wing pigmentation, we investigated the roles of two putative melanin synthesis genes, yellow and tan, using CRISPR/Cas9-mediated genome editing. We established multiple mutant strains with lesions in either gene and found that both genes were essential for normal pigmentation intensity in wing spots, though the patterns themselves persisted. Double mutants showed further reduction in pigmentation, indicating additive effects but not complete loss of patterning. Ectopic expression of wingless failed to induce normal pigmentation in yellow or tan mutants, demonstrating that both genes act downstream of wingless and are required for its pigmentation-inducing function. Furthermore, mosaic phenotypes in G0 individuals revealed quasi-cell-autonomous functions of tan, suggesting that pigmentation in D. guttifera wings depends on local availability of precursors rather than solely on transport via wing veins. This study establishes D. guttifera as a genetically tractable system for functional analyses and contributes to understanding the molecular basis of insect color pattern formation.

A Florida strain of D. guttifera (stock no.15130–1971.10, the Drosophila Species Stock Center at University of California, San Diego) was used for the study. We utilized a combination of crRNA and tracrRNA (Genome Craft type CT, Fasmac, Astugi, Japan). crRNAs were designed based on the genome sequence of a wild-type inbred line, A5 (Fukutomi et al., 2021), with Cas9 Target Finder (Kondo and Ueda 2013; http://www.shigen.nig.ac.jp/fly/nigfly/cas9) based on the following criteria: 20 nt-long and high GC content (> 66%) in 6 nt adjacent to PAM (NGG) sequence (Ren et al. 2014). A mixture of two gene-specific crRNAs (final concentration 0.3–0.5 µg/µL each), universal tracrRNA (final concentration 1–2 µg/µL) and Cas9 enzyme (final concentration 2 µg/µL, GeneArt Platinum or TrueCut Cas9 v2, ThermoFisher) was injected into embryos of the Florida strain of D. guttifera.

Phenotypes of emerged adults (G0) were observed, especially focusing on mosaic phenotypes of pigmentation. All G0 adults were crossed with wild-type Florida strain adults. In the F1 generation, mutant phenotypes were observed in some males but not in females, indicating that both yellow and tan alleles are recessive and X-linked in D. guttifera, which is also true in D. melanogaster. Mutant F1 males were crossed with the corresponding F1 females, which are phenotypically wild type but expected to carry mutant alleles as heterozygotes. The mutant strains were established by crossing mutant F2 males and mutant F2 females.

To compare the amount of black pigment among strains, we measured optical density (OD) inside/outside of spots (Fukutomi et al. 2017, 2018) of adult flies 72 ± 3 hours after eclosion. To normalize measurements, a stepped density filter (Edmund Optics, Barrington, NJ, USA) was photographed in each session and calibrated using the Rodbard function in ImageJ software (https://imagej.net/ij/). In an image of 3136 x 2352 pixels, a circle of 100 pixels diameter adjacent to the most distal companiform sensillum was used for measurement inside a spot (“Point 1”). For measurement outside of spots, a crossing point of two lines was defined as “Point 2”. One of the lines connected the posterior-distal tip of the third longitudinal vein and the anterior-distal end of the posterior cross vein. The other line connected the posterior-distal end of the fourth longitudinal vein and the leg of the perpendicular line drawn from the posterior-distal end of the second longitudinal vein to the posterior line of the third longitudinal vein. A circle of 100 pixels diameter was placed at the crossing point of these lines (“Point 2”). All statistical analyses and data visualizations were performed using R version 4.4.0 (R Core Team, 2024) with RStudio (Posit Software, PBC), and figures were generated with the ggplot2 package (Wickham, 2016). One-way ANOVA with Tukey’s HSD post hoc test was used to assess statistical significance.

To investigate the relationships between the pigmentation genes and the wingless gene, we crossed the mutant strains with a UAS-wingless (UAS-wg) strain. This UAS-wg strain has ectopic expression of wingless and associated ectopic pigmentation, probably due to an enhancer trap effect (Werner et al. 2010). We crossed the yellow¹ (y¹) strain with the UAS-wg strain, and the tan² (t²) strain with the UAS-wg strain, and observed the phenotypes of the F1 male individuals. We also measured pigmentation at Point 3, which is the location where ectopic pigmentation occurs in the original UAS-wg strain. Point 3 was defined as a crossing point of the posterior line of the third longitudinal vein and a line connecting the posterior-distal end of the second longitudinal vein and the depression at the anterior end where the fourth longitudinal vein contacts the posterior crossvein (Point C in Fukutomi et al. 2017, 2018).