Drosophila 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 tan gene contributes to pigmentation differences between two members of the virilis group: Drosophila novamexicana, which has a light yellow body color, and D. americana, which has a dark brown body color. Quantitative trait locus (QTL) mapping and expression analysis has suggested that divergence of the ebony 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 ebony null mutants in D. americana and D. novamexicana and then using reciprocal hemizygosity testing to compare the effects of each species’ ebony allele on pigmentation. We find that divergence of ebony 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 D. melanogaster, we also used the ebony null mutants to test for effects of ebony on cuticular hydrocarbon (CHC) profiles. We found that ebony 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 D. americana and D. novamexicana, such as white mutants of both species and yellow mutants in D. novamexicana, 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.

中文翻译：

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乌木影响 Drosophila americana 和 D. novamexicana 的色素沉着差异和角质层碳氢化合物

果蝇色素沉着是一个富有成效的模型系统，用于理解表型进化的遗传和发育机制。例如，之前的研究表明，tan 基因的差异导致了 virilis 组的两个成员之间的色素沉着差异：具有浅黄色体色的 Drosophila novamexicana 和具有深棕色体色的 D. americana。数量性状基因座 (QTL) 定位和表达分析表明，乌木基因的差异也可能导致这两个物种之间的色素沉着差异。在这里，我们通过使用 CRISPR/Cas9 基因组编辑在 D. americana 和 D. novamexicana 中生成乌木无效突变体，然后使用倒数半合子检验来比较每个物种的乌木等位基因对色素沉着的影响，从而直接检验这一假设。我们发现乌木的差异确实有助于物种之间的色素沉着差异，对整体体色以及背腹部中线的色素沉着差异都有影响。受近期黑腹果蝇研究的启发，我们还使用乌木无效突变体来测试乌木对角质层碳氢化合物 (CHC) 分布的影响。我们发现乌木会影响这两个物种的 CHC 丰度，但不会导致这两个物种之间 CHC 分布的质量差异。与 D. americana 和 D. novamexicana 一起工作的其他转基因资源，例如这两个物种的白色突变体和 D. novamexicana 中的黄色突变体，在这项工作的过程中产生并进行了描述。综合起来，