Disruption of a BTB-ZF transcription factor causes female sterility and melanization in the larval body of the silkworm,<i>Bombyx mori</i>

<jats:title>Abstract</jats:title><jats:p>The<jats:italic>dilute black</jats:italic>(<jats:italic>bd</jats:italic>) of the silkworm<jats:italic>Bombyx mori</jats:italic>is a recessive mutant that produces a grayish-black color in the larval integument, instead of the characteristic white color found in wild-type larvae. In addition, eggs produced by<jats:italic>bd</jats:italic>females are sterile due to a deficiency in the micropylar apparatus. We identified candidate genes responsible for the<jats:italic>bd</jats:italic>phenotype using publicly available RNA-seq data. One of these candidate genes was homologous to the<jats:italic>maternal gene required for meiosis</jats:italic>(<jats:italic>mamo</jats:italic>) of<jats:italic>Drosophila melanogaster</jats:italic>, which encodes a broad-complex, tramtrack, and bric-à-brac-zinc finger (BTB-ZF) transcription factor essential for female fertility. In three independent<jats:italic>bd</jats:italic>strains, the expression of the<jats:italic>B. mori mamo</jats:italic>(<jats:italic>Bmmamo</jats:italic>) was downregulated in the larval integument. Using a CRISPR/Cas9-mediated knockout strategy, we found that<jats:italic>Bmmamo</jats:italic>knockout mutants exhibit a grayish-black color in the larval integument and female infertility. Moreover, larvae obtained from the complementation cross between<jats:italic>bd/+</jats:italic>mutants and heterozygous knockouts for the<jats:italic>Bmmamo</jats:italic>also exhibited a grayish-black color, indicating that<jats:italic>Bmmamo</jats:italic>is responsible for the<jats:italic>bd</jats:italic>phenotype. Gene expression analysis using<jats:italic>Bmmamo</jats:italic>knockout mutants suggested that the BmMamo protein suppresses the expression of melanin synthesis genes. Previous comparative genome analysis revealed that the<jats:italic>Bmmamo</jats:italic>was selected during silkworm domestication, and we found that<jats:italic>Bmmamo</jats:italic>expression in the larval integument is higher in<jats:italic>B. mori</jats:italic>than in the wild silkworm<jats:italic>B. mandarina</jats:italic>, suggesting that the<jats:italic>Bmmamo</jats:italic>is involved in domestication-associated pigmentation changes of the silkworm.</jats:p><jats:sec><jats:title>Graphical Abstract</jats:title><jats:fig id="ufig1" position="float" orientation="portrait" fig-type="figure"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="535244v2_ufig1" position="float" orientation="portrait" /></jats:fig></jats:sec>