Female silkworms have the sex factor

Over the past 80 years, a growing body of evidence1, 2 has suggested that, in the silkworm Bombyx mori, sex is determined by the presence or absence of a maternally inherited sex chromosome, known as W. But despite years of searching for a protein-coding gene on W that could be the primary trigger for development as a female, the molecular mechanism of sex determination in the silkworm has remained unknown3. On page 633 of this issue, Kiuchi et al.4 report the discovery of a surprising, RNA-mediated pathway of sex determination in B. mori. In many insects, the development of sexual characteristics involves an evolutionarily conserved mechanism: sex-specific regulation of the gene doublesex (ref. 5). In B. mori, as in the fruit fly Drosophila melanogaster, transcription of doublesex produces different forms of messenger RNA in males and females6, a process called alternative splicing. The different mRNA transcripts then turn on sex-specific gene expression, giving rise to male or female traits5. But what is the sex-determining factor that controls splicing of Bombyx mori doublesex (Bmdsx)? In silkworms, as in most members of Lepidoptera (moths and butterflies), sex is dependent on 'WZ' sex chromosomes, in contrast to the XY system found in many other species, including mammals. In WZ systems, an embryo with one W and one Z chromosome develops as a female, and an embryo with two Z chromosomes develops as a male. The W chromosome of B. mori lacks protein-coding genes and consists mainly of transposons — mobile genetic elements that move around within the genome and can thus cause mutations. The only transcripts produced from the W chromosome are PIWI-interacting RNAs (piRNAs), small RNAs that inhibit transposons in animals' gonads by interacting with PIWI proteins7. To identify potential sex determinants on W, Kiuchi et al. performed in-depth sequencing of the RNA transcripts expressed in female and male B. mori embryos. They identified one transcript that is expressed in females at all stages of development, but never in males. This transcript is a precursor of a female-specific piRNA, which the researchers name Fem piRNA. The authors report that inhibiting the expression of Fem piRNA in female embryos changes the splicing pattern of Bmdsx mRNA from the female to the male form, suggesting that this piRNA is required for female sex determination. Furthermore, they demonstrate that Fem piRNA targets and cleaves an mRNA molecule transcribed from a gene on the Z chromosome, Masculinizer (Masc), which encodes the zinc-finger protein masculinizer (Masc). Kiuchi and colleagues find that, in the absence of Fem piRNA (in embryos lacking the W chromosome), Masc promotes male-specific splicing of Bmdsx, resulting in male development. Fem piRNA inhibits male development in WZ embryos by downregulating the level of Masc (Fig. 1). Female-specific Bmdsx splicing then occurs by default, ensuring female development. Thus, the W-encoded Fem piRNA is the long-sought primary trigger of female development. The detailed molecular mechanism revealed by this work may shed light on sex determination in most lepidopterans. Exceptions to the WZ system include 'primitive' moths such as the Micropterigidae and Hepialidae (which arose before the evolution of W and so lack this chromosome and have a Z/ZZ system like their closest relatives, caddisflies) and some 'advanced' species, such as the Eri silkworm Samia cynthia ricini, in which the W chromosome has been lost8. Because W chromosomes are transmitted only through females, they cannot undergo recombination — a mechanism that generates genetic variation in other chromosome pairs, and that also prevents mutations that negatively affect gene function from becoming fixed in the population. As a result, W chromosomes in different species have common features, including extensive genetic erosion, accumulation of repetitive DNA sequences and uniformity in DNA composition8. Why female genomes in most lepidopterans still have these chromosomes, despite their apparent uselessness and lack of protein-coding genes, might therefore seem unclear. The piRNA-driven pathway discovered by Kiuchi et al.4 could provide a rationale for their existence. How, then, is sex determined in lepidopterans that lack a W chromosome? Kiuchi and colleagues point out that the occurrence of genes equivalent to Masc in several other lepidopteran species may indicate an evolutionarily conserved role for Masc in sex determination. In such cases, a Z-counting mechanism might control sex, with a male-promoting gene such as Masc on the Z chromosome acting against a female-promoting gene on another chromosome — expression of Masc from two Z chromosomes being required to tip the balance in favour of male development. Such an ancestral Z-counting mechanism might have been re-established in species that lost the W chromosome at a later date. The only weakness in Kiuchi and co-workers' data is the absence of a conclusive assay showing that inhibition of either Fem piRNA or Masc expression can cause a change of sex from that expected from an embryo's chromosomal make-up. An inhibitor of Fem piRNA injected into WZ silkworm embryos proved to be insufficiently effective to cause male development, and genetic inhibition of Masc caused ZZ embryos to die before hatching. Nevertheless, when conducting RNA sequence analysis of the Masc-inhibited ZZ embryos before death, the authors made another observation — the Masc protein is required for dosage compensation, the process by which the expression of genes on the Z chromosome is downregulated in ZZ embryos to lower the genes' transcription to the same level as in ZW embryos. Dosage compensation has not previously been reported in lepidopterans. Kiuchi and colleagues' evidence that this occurs in B. mori contradicts results showing8 that male lepidopterans tolerate a double dose of Z-linked gene transcripts compared with females, but is consistent with data9 on global gene expression in the silkworm indicating that gene expression from the Z chromosome occurs at the same level in males and females. As the authors note, the lethal effect of Masc inhibition is similar to the 'male-killing action' of the bacterium Wolbachia in several lepidopteran species: infection by Wolbachia kills male embryos10. It seems that this, too, may result from a failure of dosage compensation. This is thought to be the first example of a sex-determining pathway controlled by the presence or absence of a piRNA. Whether this pathway is evolutionarily conserved in lepidopterans will certainly be the subject of future research. Kiuchi and co-workers' findings also pave the way to understanding the molecular mechanism by which Wolbachia and other endosymbiotic bacteria manipulate the sex of their host. Finally, the results open up options for artificial sex control in silkworms (for example, to promote the development of males, which produce more silk than do females2) and possible ways to control lepidopteran pests. Download references