Identification and functional analysis of sex-determining genes in the spongy moth, Lymantria dispar (Lepidoptera: Erebidae)

The spongy moth (Lymantria dispar) employs a female heterogametic sex-determination system, where the female sex-determining factor (F factor) is located on the W chromosome, and the male sex-determining factor (M factor) is located on the Z chromosome. The sex-determining capabilities of the F factor and M factor vary among subspecies. Consequently, L. dispar serves as an excellent model for studying the mechanisms underlying the evolution and diversity of sex-determining genes. However, the genes encoding the F and M factors, as well as the molecular functions of their translation products, remain unidentified. In this study, we identified a L. dispar Masculinizer ortholog (LdMasc) and found that this gene is highly expressed in male embryos during the sex-determination stage. When LdMasc expression was silenced using embryonic RNA interference (RNAi), the expression pattern of L. dispar doublesex (Lddsx), the master regulatory gene for sex differentiation, shifted from the male-specific form to the female-specific form in male embryos. To identify potential F factors, we screened for genes that were exclusively expressed in females across multiple tissues and located only within the female genome. This screening yielded four unigenes with sequences displaying high homology to each other. These unigenes formed a tandem repeat, comprising approximately 100 copies within a 200 kbp region of the W chromosome-derived contig. We designated these unigenes as Fet-W (female-specifically expressed transcript from the W chromosome). RT-PCR analysis revealed that Fet-W was expressed in a female-specific manner during the sex-determination stage. Suppression of Fet-W expression by embryonic RNAi led to an increase in LdMasc expression in females and a corresponding shift in dsx expression patterns from the female-specific to the male-specific form. These findings strongly suggest that the F factor in L. dispar is Fet-W, whereas the M factor is LdMasc.