Various organisms have color patterns on their body surfaces, and these color patterns are thought to contribute to physiological regulation, communication with conspecifics, and signaling with the environment. An adult fly of Drosophila guttifera (Insecta: Diptera: Drosophilidae) has melanin pigmentation patterns on its body and wings. Though D. guttifera has been used for research into color pattern formation, how its pupal development proceeds and when the pigmentation starts have not been well studied. In this study, we defined the pupal stages of D. guttifera and measured the pigment content of wing spots from the pupal period to the period after eclosion. Using a transgenic line which carries eGFP connected with an enhancer of yellow, a gene necessary for melanin synthesis, we analyzed the timing at which the yellow enhancer starts to drive eGFP. We also analyzed the distribution of Yellow-producing cells, as indicated by the expression of eGFP during pupal and young adult periods. The results suggested that Yellow-producing cells were removed from wings within 3 h after eclosion, and wing pigmentation continued without epithelial cells. Furthermore, the results of vein cutting experiments showed that the transport of melanin precursors through veins was necessary for wing pigmentation. These results showed the importance of melanin precursors transported through veins and of extracellular factors which were secreted from epithelial cells and left in the cuticle.

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

A polka-dotted fruit fly, Drosophila guttifera, has a unique pigmentation pattern made of black melanin and serves as a good model system to study color pattern formation. Because of its short generation time and the availability of transgenics, it is suitable for dissecting the genetic mechanisms of color pattern formation. While the ecology and life history of D. guttifera in the wild are not well understood, it is known to be resistant to a mushroom toxin, and this physiological trait is under molecular scrutiny. Pigmentation around crossveins and longitudinal vein tips is common in closely related species of the quinaria group, in addition to which D. guttifera has evolved species-specific pigmentation spots around the campaniform sensilla. Regulatory evolution of the Wnt signaling ligand Wingless, which locally induces pigmentation in the developing wing epithelium, has driven the evolution of distinct aspects of wing and body pigmentation. A melanin biosynthesis pathway gene, yellow, is also involved in the elaboration of these traits, downstream of wingless. Unraveling the detailed mechanism of pigmentation pattern formation of this species sheds light on the general principles of morphological evolution and foreshadows potential parallels with other systems, such as the pigmented wings of butterflies.

How evolutionary novelties have arisen is one of the central questions in evolutionary biology. Preexisting gene regulatory networks or signaling pathways have been shown to be co‐opted for building novel traits in several organisms. However, the structure of entire gene regulatory networks and evolutionary events of gene co‐option for emergence of a novel trait are poorly understood. In this study, to explore the genetic and molecular bases of the novel wing pigmentation pattern of a polka‐dotted fruit fly (Drosophila guttifera), we performed de novo genome sequencing and transcriptome analyses. As a result, we comprehensively identified the genes associated with the pigmentation pattern. Furthermore, we revealed that 151 of these associated genes were positively or negatively regulated by wingless, a master regulator of wing pigmentation. Genes for neural development, Wnt signaling, Dpp signaling, and effectors (such as enzymes) for melanin pigmentation were included among these 151 genes. None of the known regulatory genes that regulate pigmentation pattern formation in other fruit fly species were included. Our results suggest that the novel pigmentation pattern of a polka‐dotted fruit fly might have emerged through multistep co‐options of multiple gene regulatory networks, signaling pathways, and effector genes, rather than recruitment of one large gene circuit.

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