Lepidopteran sex determination: a cascade of surprises.

Sex determination is a developmental pathway that fixes the sexual fate (male or female) of an individual at early stages of embryonic development. This pathway is ideally suited for evolutionary studies given the astoundingly diverse mechanisms found in the animal kingdom. In particular, insects use multiple different cues to specify the sexual fate of an individual. In this review, we focus on genes and genetic interactions involved in the sex determination of insect species belonging to the order Lepidoptera. Unique features of the lepidopteran sex determination system are discussed.

Doublesex: a conserved downstream gene controlled by diverse upstream regulators.

Sex determination, an integral precursor to sexual reproduction, is required to generate morphologically distinct sexes. The molecular components of sex-determination pathways regulating sexual differentiation have been identified and characterized in different organisms. The Drosophila doublesex (dsx) gene at the bottom of the sex-determination cascade is the best characterized candidate so far, and is conserved from worms (mab3 of Caenorhabditis elegans) to mammals (Dmrt-1). Studies of dsx homologues from insect species belonging to different orders position them at the bottom of their sex-determination cascade. The dsx homologues are regulated by a series of upstream regulators that show amazing diversity in different insect species. These results support the Wilkin's hypothesis that evolution of the sex-determination cascade has taken place in reverse order, the bottom most gene being most conserved and the upstream genes having been recruited at different times during evolution. The pre-mRNA of dsx is sex-specifically spliced to encode male or female-specific transcription factors that play an important role in the regulation of sexually dimorphic characters in different insect species. The generalization that dsx is required for somatic sexual differentiation culminated with its functional analysis through transgenesis and knockdown experiments in diverse species of insects. This brief review will focus on the similarities and variations of dsx homologues that have been investigated in insects to date.

Two female-specific DSX proteins are encoded by the sex-specific transcripts of dsx, and are required for female sexual differentiation in two wild silkmoth species, Antheraea assama and Antheraea mylitta (Lepidoptera, Saturniidae).

doublesex (dsx) is the bottom most gene of the sex-determination cascade of Drosophila melanogaster. The pre-mRNA of dsx splices to produce male- and female-specific transcripts which code for the male- and female-specific proteins, respectively. dsx homologues have been characterized from different (many in Diptera, two in Hypmenoptera and only one in Lepidoptera) insect species. Sex-specific splice forms of dsx pre-mRNA in all these species code for one male- and one female-specific DSX proteins, which regulate the downstream target genes responsible for sex-specific characters. In the present study we have cloned and characterized the dsx homologues from two saturniid silkmoths, Antheraea assama and Antheraea mylitta. The divergence time between Saturniidae and Bombycidae to which the domesticated silkworm, Bombyx mori belongs is estimated to be around 160.9 MY. Interestingly, the dsx pre-mRNA of these wild silkmoths sex-specifically splices to generate multiple splice variants. On the basis of their open reading frame (ORF) and conceptual translation, two female-specific (DSX(F1) and DSX(F2)) and one male-specific (DSX(M)) proteins could be inferred, in both the moths. Presence or absence of a 15 bp stretch within the ORF of the two groups of female-specific transcripts resulted in the production of two distinct female-specific DSX proteins. The sex-specific DSX proteins have common amino-terminal sequence but sex-specific carboxy termini. The two female-specific DSX proteins (DSX(F1) and DSX(F2)) share common DNA binding domain (DM domain) and oligomerization domain (OD domain) and differ only at their extreme C-termini by 21aa. Functional analysis of dsx transcripts in A. assama by dsRNA mediated knock-down resulted in complete abolition of expression of vitellogenin and hexamerin genes, the direct targets of the DSX proteins, irregular differentiation of gonads, and drastic reduction in fecundity and hatchability. Together, these results suggest the involvement of both the female-specific DSX proteins in the process of female sexual differentiation. Further, conservation of the 4th exon sequence, especially the PESS sequence responsible for the sex-specific splicing of Bmdsx in the female-specific transcripts of Aadsx and Amydsx, indicated the existence of a common mechanism of sex-specific splicing of dsx homologues in silkmoths. To our knowledge this is the first report of existence of multiple splice forms of dsx pre-mRNA encoding two female-specific DSX proteins.