Allele-specific knockouts reveal a role for apontic-like in the evolutionary loss of larval melanin pigmentation in the domesticated silkworm, Bombyx mori.

The domesticated silkworm, Bombyx mori, and its wild progenitor, B. mandarina, are extensively studied as a model case of the evolutionary process of domestication. A conspicuous difference between these species is the dramatic reduction in melanin pigmentation in both larval and adult B. mori. Here we evaluate the efficiency of CRISPR/Cas9-targeted knockouts of pigment-related genes as a tool to understand their potential contributions to domestication-associated melanin pigmentation loss in B. mori. To demonstrate the efficacy of targeted knockouts in B. mandarina, we generated a homozygous CRISPR/Cas9-targeted knockout of yellow-y. In yellow-y knockout mutants, black body colour became lighter throughout the larval, pupal and adult stages, confirming a role for this gene in melanin pigment formation. Further, we performed allele-specific CRISPR/Cas9-targeted knockouts of the pigment-related transcription factor, apontic-like (apt-like) in B. mori × B. mandarina F hybrid individuals which exhibit B. mandarina-like larval pigmentation. Knockout of the B. mandarina allele of apt-like in F embryos results in white patches on the dorsal integument of larvae, whereas corresponding knockouts of the B. mori allele consistently exhibit normal F larval pigmentation. These results demonstrate a contribution of apt-like to the evolution of reduced melanin pigmentation in B. mori. Together, our results demonstrate the feasibility of CRISPR/Cas9-targeted knockouts as a tool for understanding the genetic basis of traits associated with B. mori domestication.