Abstract

Recent identification of a Piwi-interacting RNA (piRNA)-initiated sex determination cascade in the silkworm, Bombyx mori , provides novel insights into high diversity of insect sex determination pathways. In this system, the W-chromosome-derived Fem piRNA is the primary sex determination signal. A CCCH-type zinc finger gene Masculinizer ( Masc ), which is targeted by Fem piRNA-PIWI complex in female animals, is indispensable for male-specific splicing of B. mori doublesex ( Bmdsx ). Although many genes involved in this cascade have been identified, the regulatory mechanisms of silkworm sex determination remain to be elucidated. Here we show that another CCCH-type zinc finger gene, Bmznf-2 , is a masculinization factor in B. mori. Bmznf-2 shows testis-abundant expression and loss of Bmznf-2 function via clustered regularly interspaced short palindromic repeats / single-guide RNA-mediated mutagenesis results in feminized differentiation and appearance of the female-specific splicing variants of Bmdsx transcripts in males. In contrast, there is no phenotypic consequence in mutant females. In mutant males, relative messenger RNA expression levels of female-dominant genes such as vitellogenin and sex-specific storage protein 1 are significantly elevated while several male-dominant genes are significantly down-regulated. Furthermore, male mutants show delayed developmental timing, smaller body sizes of larvae and malformation of moth wings. Our data thus reveal that Bmznf-2 plays an indispensable role in silkworm male sexual differentiation.