Functional analysis of sex-determination genes by gene silencing with LNA–DNA gapmers in the silkworm, Bombyx mori

•The anti-sense LNA–DNA gapmers efficiently knockdown Fem and ImpM expressions.•Masc knockdown dramatically reduces ImpM expression in male embryos.•We herein propose a possible genetic cascade regulating sex determination in B. mori.

The sexual fate of B. mori is determined genetically; ZW, female and ZZ, male. Recently, we successfully identified a strong candidate gene at the top of the sex determination cascade in B. mori. This gene was termed Feminizer (Fem) and revealed to be a source of Fem-piRNA. Further, we found that B. mori doublesex (Bmdsx) splicing was markedly altered to produce the male-type isoform when a Fem-piRNA inhibitor was injected into ZW embryos. Moreover, knockdown of Masculinizer (Masc), a Fem-piRNA target gene, altered to produce the female-type isoform of Bmdsx in male embryos. However, it remains unclear as to whether Masc directly regulates the sex-specific expression of Bmdsx. In previous studies, we determined that the male-specific isoform of the Bombyx homolog of IGF-II mRNA-binding protein (ImpM) was involved in the male-specific splicing of Bmdsx. In an attempt to clarify the genetic relationship between Fem, Masc, ImpM, and Bmdsx, knockdown experiments were performed. Knockdown of Fem shifted into male-type Bmdsx, ImpM and Masc in female embryos. Knockdown of Masc led to the production of the female-type Bmdsx and a dramatic reduction in ImpM expression in male embryos. Knockdown of ImpM shifted Bmdsx splice mode from the male-type into the female-type. Our results suggest that: (1) Fem reduces Masc expression, (2) Masc dramatically induces ImpM expression, and (3) ImpM shifting Bmdsx splice mode from the female-type into the male-type. Based on these findings, we propose a possible genetic cascade regulating sex determination in B. mori.

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