Masculinizer is not post-transcriptionally regulated by female-specific piRNAs during sex determination in the Asian corn borer, Ostrinia furnacalis

Lepidopteran insects are heterogametic in females, although most insect species are heterogametic in males. In a lepidopteran model species, the silkworm Bombyx mori (Bombycoidea), the uppermost sex determinant Feminizer (Fem) has been identified on the female-specific W chromosome. Fem is a precursor of PIWI-interacting small RNA (piRNA). Fem piRNA forms a complex with Siwi, one of the two B. mori PIWI-clade Argonaute proteins. In female embryos, Fem piRNA-Siwi complex cleaves the mRNA of the male-determining gene Masculinizer (Masc), directing the female-determining pathway. In male embryos, Masc activates the male-determining pathway in the absence of Fem piRNA. Recently, W chromosome-derived piRNAs complementary to Masc mRNA have also been identified in the diamondback moth Plutella xylostella (Yponomeutoidea), indicating the convergent evo-lution of piRNA-dependent sex determination in Lepidoptera. Here, we show that this is not the case in the Asian corn borer, Ostrinia furnacalis (Pyraloidea). Although our previous studies demonstrated that O. furnacalis Masc (OfMasc) has a masculinizing function in the embryonic stage, the expression level of OfMasc was indistin-guishable between the sexes at the timing of sex determination. Deep sequencing analysis identified no female -specific small RNAs mapped onto OfMasc mRNA. Embryonic knockdown of two PIWI genes did not affect the expression level of OfMasc in either sex. These results demonstrated that piRNA-dependent reduction of Masc mRNA in female embryos is not a common strategy of sex determination, which suggests the possibility of divergent evolution of sex determinants across the order Lepidoptera.

Automated summary

Lepidopteran insects show a different mechanism of sex determination compared to most insect species. The silkworm Bombyx mori, a model species in this group, has a sex-determining gene called Feminizer (Fem) on the female-specific W chromosome. Fem is a precursor of PIWI-interacting small RNA (piRNA) that cleaves the mRNA of the male-determining gene Masc, leading to female development. However, this piRNA-dependent sex determination strategy is not observed in the Asian corn borer, Ostrinia furnacalis. Despite previous findings of masculinizing function of the Masc gene (OfMasc) in O. furnacalis embryos, there was no difference in the expression level of OfMasc between males and females during sex determination. Furthermore, deep sequencing analysis did not identify any female-specific small RNAs mapped onto OfMasc mRNA. Knockdown of two PIWI genes also did not affect the expression level of OfMasc in either sex. These findings suggest the possibility of divergent evolution of sex determinants in Lepidoptera.