Hemimetabolous insects elucidate the origin of sexual development via alternative splicing

Insects are the only animals in which sexual differentiationis controlled by sex-specific RNA splicing. The doublesex(dsx) transcription factor produces distinct male and female protein isoforms(DsxM and DsxF) under the control of the RNA splicingfactor transformer (tra). tra itself is also alternatively splicedso that a functional Tra protein is only present in females; thus, DsxM is produced by default, while DsxF expression requires Tra. The sex-specific Dsx isoforms are essential for both male and female sexual differentiation. This pathway is profoundly different from the molecular mechanisms that control sex-specific development in other animal groups. In animals as different as vertebrates, nematodes, and crustaceans, sexual differentiationinvolves male-specific transcription of dsx-related transcription factors that are not alternatively splicedand play no role in female sexual development. To understand how the unique splicing-based mode of sexual differentiationfound in insects evolved from a more ancestral transcription-based mechanism, we examined dsx and tra expression in three basal, hemimetabolousinsect orders. We find that functional Tra protein is limited to females in the kissingbug Rhodniusprolixus (Hemiptera), but is present in both sexes in the louse Pediculus humanus(Phthiraptera) and the cockroachBlattella germanica ( Blattodea). Although alternatively spliceddsx isoforms are seen in all these insects, they are sex-specific in the cockroachand the kissingbug but not in the louse. In B. germanica, RNAi experiments show that dsx is necessary for male, but not female, sexual differentiation, while tra controls female development via a dsx-independent pathway. Our results suggest that the distinctive insect mechanism based on the tra-dsx splicingcascade evolved in a gradual, mosaic process: sex-specific splicingof dsx predates its role in female sexual differentiation, while the role of tra in regulating dsx splicingand in sexual development more generally predates sex-specific expression of the Tra protein. We present a model where the canonical tra-dsx axis originated via merger between expanding dsx function (from males to both sexes) and narrowing tra function (from a general splicing factorto the dedicated regulator of dsx).