Affects Pigmentation Divergence and Cuticular Hydrocarbons in and

pigmentation has been a fruitful model system for understanding the genetic and developmental mechanisms underlying phenotypic evolution. For example, prior work has shown that divergence of the gene contributes to pigmentation differences between two members of the virilis group: , which has a light yellow body color, and , which has a dark brown body color. Quantitative trait locus (QTL) mapping and expression analysis has suggested that divergence of the gene might also contribute to pigmentation differences between these two species. Here, we directly test this hypothesis by using CRISPR/Cas9 genome editing to generate null mutants in and and then using reciprocal hemizygosity testing to compare the effects of each species' allele on pigmentation. We find that divergence of does indeed contribute to the pigmentation divergence between species, with effects on both the overall body color as well as a difference in pigmentation along the dorsal abdominal midline. Motivated by recent work in , we also used the null mutants to test for effects of on cuticular hydrocarbon (CHC) profiles. We found that affects CHC abundance in both species, but does not contribute to qualitative differences in the CHC profiles between these two species. Additional transgenic resources for working with and such as mutants of both species and mutants in , were generated in the course of this work and are also described. Taken together, this study advances our understanding of loci contributing to phenotypic divergence and illustrates how the latest genome editing tools can be used for functional testing in non-model species.