Injecting <em>Gryllus bimaculatus</em> Eggs

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been ...

National University of Singapore

Despite the variety, prominence, and adaptive significance of butterfly wing patterns, surprisingly little is known about the genetic basis of wing color diversity. Even though there is intense interest in wing pattern evolution and development, the technical challenge of genetically manipulating butterflies has slowed efforts to functionally characterize color pattern development genes. To identify candidate wing pigmentation genes, we used RNA sequencing to characterize transcription across multiple stages of butterfly wing development, and between different color pattern elements, in the painted lady butterfly  This allowed us to pinpoint genes specifically associated with red and black pigment patterns. To test the functions of a subset of genes associated with presumptive melanin pigmentation, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing in four different butterfly genera. , , and  knockouts displayed reduction of melanin pigmentation, consistent with previous findings in other insects. Interestingly, however, , , and  knockouts revealed that these genes have localized effects on tuning the color of red, brown, and ochre pattern elements. These results point to previously undescribed mechanisms for modulating the color of specific wing pattern elements in butterflies, and provide an expanded portrait of the insect melanin pathway.

Genetic Basis of Melanin Pigmentation in Butterfly Wings.

The cuticular skeleton of a butterfly wing scale cell is an exquisitely finely sculpted material that can contain pigments, produce structural colors, or both. While cuticle rigidity and pigmentation depend on the products of the melanin pathway, little is known about whether genes in this pathway also play a role in the development of specific scale morphologies. Here, we use CRISPR/Cas9 to show that knockout mutations in five genes that function in the melanin pathway affect both the fine structure and the coloration of the wing scales. Most dramatically, mutations in the yellow gene lead to extra horizontal laminae on the surface of scales, whereas mutations in DDC gene lead to taller and sheet-like vertical laminae throughout each scale. We identify genes affecting the development of color and scale morphology, the regulation and pleiotropic effects of which may be important in creating and limiting the diversity of the structural and pigmentary colors observed in butterflies.

Yuji Matsuoka

Department of Biological Sciences

National University of Singapore

Injecting Gryllus bimaculatus Eggs