Two of the three Transformer-2 genes are required for ovarian development in Aedes albopictus

In Drosophila melanogaster, transformer-2 (tra2) plays an essential role in the sex-specific splicing of doublesex (dsx) and fruitless (fru), two key transcription factor genes that program sexual differentiation and regulate sexual behavior. In the present study, the sequences and expression profiles of three tra2 (Aalbtra2) genes in the Asian tiger mosquito, Aedes albopictus (Ae. albopictus) were characterized. Phylogenetic analysis revealed that these paralogs resulted from two duplication events. The first occurred in the common ancestor of Culicidae, giving rise to the tra2-α and tra2-β clades that are found across divergent mosquito genera, including Aedes, Culex, and Anopheles. The second occurred within the tra2-α clade, giving rise to tra2-γ in Ae. albopictus. In addition to the conserved RNA recognition motif (RRM), arginine-rich/serine-rich regions (RS domains) and a linker region, a glycine-rich region located between the RRM and RS2 was observed in Tra2-α and Tra2-γ of Ae. albopictus that has not yet been described in the Tra2 proteins of dipteran insects. Quantitative real-time PCR detected relatively high levels of transcripts from all three tra2 paralogs in 0-2 h embryos, suggesting maternal deposition of these transcripts. All three Aalbtra2 genes were highly expressed in the ovary, while Aalbtra2-β was also highly expressed in the testis. RNAi-mediated knockdown of any or all Aalbtra2 genes did not result in an obvious switch of the sex-specificity in dsx and fru splicing in the whole-body samples. However, knockdown of transcripts from all three tra2 genes significantly reduced the female isoform of dsx mRNA and increased the male isoform of the dsx mRNA in both the ovary and the fat body in adult females. Furthermore, knockdown of either Aalbtra2-α or Aalbtra2-γ or all three Aalbtra2 led to a decrease in ovariole number and ovary size after a blood meal. Taken together, these results indicate that two of the three tra2 genes affect female ovarian development.