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.

中文翻译：

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CCCH 锌指基因调控家蚕双性可变剪接和雄性发育

最近鉴定的家蚕 Bombyx mori中由 Piwi 相互作用 RNA (piRNA) 启动的性别决定级联提供了对昆虫性别决定途径高度多样性的新见解。在该系统中，W 染色体衍生的Fem piRNA 是主要的性别决定信号。甲CCCH型锌指基因Masculinizer（睫毛膏），其通过靶向有限元piRNA基因-PIWI在雌性动物复杂，是不可缺少的用于雄性特异性剪接家蚕doublesex（Bmdsx）。尽管已经确定了参与该级联的许多基因，但家蚕性别决定的调控机制仍有待阐明。在这里，我们展示了另一个 CCCH 型锌指基因Bmznf-2是家蚕中的男性化因子。Bmznf-2显示睾丸丰富的表达和Bmznf-2功能的丧失，通过成簇的规则间隔短回文重复/单向导 RNA 介导的突变导致女性化分化和男性Bmdsx转录本的女性特异性剪接变体的出现。相比之下，在突变雌性中没有表型结果。在突变雄性中，雌性优势基因（如卵黄蛋白原）的相对信使 RNA 表达水平和性别特异性储存蛋白 1显着升高，而几个雄性优势基因显着下调。此外，雄性突变体表现出发育时间延迟、幼虫体型较小和蛾翅畸形。我们的数据因此表明Bmznf-2在蚕雄性分化中起着不可或缺的作用。