BACKGROUND: The primary signals of sex determination in insects are diverse and evolve erratically. This also appears to be the case with moths and butterflies. In the silkworm Bombyx mori, female development is triggered by a W chromosome-derived Feminizer (Fem) piRNA that degrades the mRNA of the Z-linked Masculinizer (Masc) gene, which controls male development. We investigated whether this mechanism is conserved in another group of Lepidoptera. RESULTS: We identified a putative feminizing piRNA and many partial copies of the EkMasc gene on the W chromosome of Ephestia kuehniella. The piRNA is generated by a repetitive W-linked sequence named E. kuehniella Moth-overruler-of-masculinization (EkMom). EkMom piRNA shows high similarity to a region of Z-linked EkMasc and is expressed at the onset of female development, but has no relationship to the B. mori Fem piRNA. We then mapped small RNA-seq data from embryos of the related Plodia interpunctella to the PiMasc gene and identified a single small RNA, a PiMom piRNA, able to target PiMasc and with high sequence identity to the EkMom piRNA. Both the PiMom and EkMom repeats are present in high copy number and form a single cluster on the W chromosome. In both species, the Mom piRNA is responsible for Masc mRNA cleavage, clearly demonstrating that the Mom piRNA triggers female development. CONCLUSIONS: Our study provides multiple lines of evidence that Mom piRNA is the primary sex-determining signal in two pyralid moths and highlights a possible pathway for the origin of feminizing piRNAs in Lepidoptera. The similarity in female sex determination between the phylogenetically distant species suggests convergent evolution of feminizing piRNAs in Lepidoptera.

Biology Centre of the Czech Academy of Sciences Institute of Entomology 370 05 České Budějovice Czech Republic

Department of Ecology and Evolutionary Biology University of Kansas Lawrence KS 66045 USA

Faculty of Medicine 2 Charles University Prague and University Hospital Motol 150 06 Prague 5 Czech Republic

Faculty of Science University of South Bohemia 370 05 České Budějovice Czech Republic

Groningen Institute for Evolutionary Life Sciences University of Groningen 9747 AG Groningen The Netherlands

Laboratory of Entomology Wageningen University and Research 6700 AA Wageningen The Netherlands

Present Address European Molecular Biology Laboratory 69117 Heidelberg Germany

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