The evolution of dosage compensation produces similar expression of sex-linked and autosomal genes in the heterogametic sex. The silkworm ( Bombyx mori ), a lepidopteran insect, has a female heterogametic WZ sex determination system. A Z-linked gene, Masculinizer ( Masc ), is the primary determinant of maleness and dosage compensation in B. mori . However, it remains unknown whether one of the two Z chromosomes is inactivated or both Z chromosomes are suppressed in B. mori males. Hence, we performed transcriptome analysis using hybrids between two B. mori strains and analysed allele-specific expression to distinguish these alternatives. Our analysis revealed that genes on both the maternal and paternal Z chromosomes are transcriptionally upregulated in Masc knocked down males. We therefore conclude that both Z chromosomes are transcriptionally downregulated in B. mori males, similar to the system in Caenorhabditis elegans .

Dosage compensation balances the expression of sex-chromosome-linked genes with autosome-linked genes in the heterogametic sex. The silkworm (Bombyx mori), a lepidopteran model insect, uses the female heterogametic WZ sex determination system. In B. mori, a Z-linked gene, Masculinizer (Masc), is the primary determinant of maleness and dosage compensation. However, it remains unknown whether one of the two Z chromosomes is inactivated or both Z chromosomes are suppressed in B. mori males. Hence, we performed transcriptome analysis using hybrids between two B. mori strains and analyzed the allele-specific expression (ASE) to solve the problem. The ASE analysis revealed that the genes located on the maternal and paternal Z chromosomes are transcriptionally upregulated in Masc knocked down males. Also, our results revealed that two Z chromosomes are transcriptionally downregulated in B. mori males as observed in Caenorhabditis elegans.

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 F1 hybrid individuals which exhibit B. mandarina‐like larval pigmentation. Knockout of the B. mandarina allele of apt‐like in F1 embryos results in white patches on the dorsal integument of larvae, whereas corresponding knockouts of the B. mori allele consistently exhibit normal F1 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.

Dosage compensation is a process that produces a similar expression of sex-linked and autosomal genes. In the silkworm Bombyx mori with a WZ sex-determination system, the expression from the single Z in WZ females matches that of ZZ males due to the suppression of Z-linked genes in males. A primary maleness determinant gene, Masculinizer (Masc), is also required for dosage compensation. In females, P-element induced wimpy testis (PIWI) is complexed with the W chromosome-derived female-specific Feminizer (Fem) PIWI-interacting RNA (piRNA) and cleaves Masc mRNA. When Fem piRNA-resistant Masc cDNA (Masc-R) is overexpressed in both sexes, only female larvae are dead during the larval stage. In this study, transcriptome analysis was performed in neonate larvae to examine the effects of Masc-R overexpression on a global gene expression profile. Z-linked genes were globally repressed in Masc-R-overexpressing females due to force-driven dosage compensation. In contrast, Masc-R overexpression had little effect on the expression of Z-linked genes and the male-specific isoform of B. mori insulin-like growth factor II mRNA-binding protein in males, indicating that excessive Masc expression strengthens neither dosage compensation nor maleness in males. Fourteen genes were differentially expressed between Masc-R-overexpressing and control neonate larvae in both sexes, suggesting Masc functions other than dosage compensation and masculinization.HighlightsTranscriptome analysis was performed in Masc-R-overexpressing neonate Bombyx mori larvae.Z-linked genes were globally suppressed in Masc-R-overexpressing females.Masc-R overexpression had little effect on BmImpM expression in males.Several genes may be controlled by Masc-R regardless of sex.

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