W chromosome-specific paralogs of the male-determining gene LdMasc exhibits a female- determining ability in the spongy moth, Lymantria dispar

Background Sex determination is a common developmental process in most organisms that exhibit sexual reproduction. Nevertheless, the modes of sex determination are highly diverse, and master sex-determining genes sometimes differ substantially even among closely related species. We explored the molecular basis underlying this striking diversity in the spongy moth, Lymantria dispar , whose master sex-determining factors exhibit continuous variation among populations. Results We previously identified the female-determining gene Fet-W and the male-determining gene LdMasc in the Japanese spongy moth ( Lymantria dispar japonica ). The number of raw RNA sequencing reads mapped to the LdMasc coding sequence region corresponding to exons 9–11 was higher in females than in males. Consistent with this finding, we identified four unigenes composed of the nucleotide sequences from LdMasc exons 9–11. Reverse-transcript polymerase chain reaction (RT-PCR) and genomic PCR using primers specific to these unigenes strongly indicated that these four unigenes originated from the W-linked gene. A nucleotide BLAST search across the entire L . d . japonica genome found at least 52 copies of nucleotide sequences nearly identical to the approximately 5.6-kb genomic sequence spanning intron 8 to exon 11 of LdMasc . Similar sequences were identified in the W chromosome contigs of Lymantria dispar dispar and Lymantria dispar asiatica , with 4 and 6 copies, respectively. We considered these sequences to be W chromosome paralogs of LdMasc and named them LdMasc-W . RT-PCR analysis demonstrated that LdMasc-W exhibits specific expression during the sex determination stage. Knockdown of LdMasc-W using embryonic RNA induced male-specific splicing of doublesex ( dsx ), a master regulatory gene for sexual development, in females. F1 hybrid females obtained by mating females of L. umbrosa , which that lacks LdMasc-W , with L. d. japonica males exhibited lethality or intersexual phenotypes. Conclusions These results strongly suggest that LdMasc-W functions to promote female determination by suppressing the expression of the male-determining gene LdMasc , similar to the female-determining gene Fem identified in other Lepidoptera species. This means that the spongy moth is a rare species possessing two W-linked female-determining genes: Fet-W and LdMasc-W .