In insect sex determination a primary signal starts the genetic sex determination cascade that, in most insect orders, is subsequently transduced down the cascade by a transformer (tra) ortholog. Only a female-specifically spliced tra mRNA yields a functional TRA-protein that forms a complex with TRA2, encoded by a transformer-2 (tra2) ortholog, to act as a sex specific splicing regulator of the downstream transcription factors doublesex (dsx) and fruitless (fru). Here, we identify the tra2 ortholog of the haplodiploid parasitoid wasp N. vitripennis (Nv-tra2) and confirm its function in N. vitripennis sex determination. Knock down of Nv-tra2 by parental RNA interference (pRNAi) results in complete sex reversal of diploid offspring from female to male, indicating the requirement of Nv-tra2 for female sex determination. As Nv-tra2 pRNAi leads to frequent lethality in early developmental stages, maternal provision of Nv-tra2 transcripts is apparently also required for another, non-sex determining function during embryogenesis. In addition, lethality following Nv-tra2 pRNAi appears more pronounced in diploid than in haploid offspring. This diploid lethal effect was also observed following Nv-tra pRNAi, which served as a positive control in our experiments. As diploid embryos from fertilized eggs have a paternal chromosome set in addition to the maternal one, this suggests that either the presence of this paternal chromosome set or the dosage effect resulting from the diploid state is incompatible with the induced male development in N. vitripennis caused by either Nv-tra2 or Nv-tra pRNAi. The role of Nv-tra2 in activating the female sex determination pathway yields more insight into the sex determination mechanism of Nasonia.

Sexually dimorphic traits in insects are rapidly evolving due to sexual selection which can ultimately lead to speciation. However, our knowledge of the underlying sex-specific molecular mechanisms is still scarce. Here we show that the highly conserved gene, Doublesex (Dsx), regulates rapidly diverging sexually dimorphic traits in the model parasitoid wasp Nasonia vitripennis (Hymenoptera: Pteromalidae). We present here the revised full Dsx gene structure with an alternative first exon, and two additional male NvDsx isoforms, which gives important insights into the evolution of the sex-specific oligomerization domains and C-termini. We show the sex-specific NvDsx expression throughout development, and demonstrate that transient NvDsx silencing in different male developmental stages dramatically shifts the morphology of two sexually dimorphic traits from male to female, with the effect being dependent on the timing of silencing. In addition, transient silencing of NvDsx in early male larvae affects male genitalia tissue growth but not morphology. This indicates that male NvDsx is actively required to suppress female-specific traits and to promote male-specific traits during specific developmental windows. These results also strongly suggest that in N. vitripennis most sex-specific tissues fully differentiate in the embryonic stage and only need the input of NvDsx for growth afterwards. This provides a first insight into the regulatory activity of Dsx in the Hymenoptera and will help to better understand the evolutionary and molecular mechanisms involved in sex-specific development in this parasitoid wasp, which can eventually lead to the development of new synthetic genetics-based tools for biological pest control by parasitoid wasps.Significance StatementIn insects, male and female differentiation is regulated by the highly conserved transcription factor Doublesex (Dsx). The role of Dsx in regulating rapidly evolving sexually dimorphic traits has received less attention, especially in wasps and bees. Here, we mainly focused on Dsx regulation of two sexually dimorphic traits and male genitalia morphology in the parasitoid wasp, Nasonia vitripennis. We demonstrate that Dsx actively regulates male-specific tissue growth and morphology during specific developmental windows. These findings will help to better understand the molecular mechanisms underlying the rapid evolution of sexual differentiation and sexually dimorphic traits in insects, but may also be the starting point for the development of new tools for biological control of pest insects by parasitoid wasps.

Sex determination, the developmental process by which sexually dimorphic phenotypes are established, evolves fast. Evolutionary turnover in a sex determination pathway may occur via selection on alleles that are genetically linked to a new master sex determining locus on a newly formed proto‐sex chromosome. Species with polygenic sex determination, in which master regulatory genes are found on multiple different proto‐sex chromosomes, are informative models to study the evolution of sex determination and sex chromosomes. House flies are such a model system, with male determining loci possible on all six chromosomes and a female‐determiner on one of the chromosomes as well. The two most common male‐determining proto‐Y chromosomes form latitudinal clines on multiple continents, suggesting that temperature variation is an important selection pressure responsible for maintaining polygenic sex determination in this species. Temperature‐dependent fitness effects could be manifested through temperature‐dependent gene expression differences across proto‐Y chromosome genotypes. These gene expression differences may be the result of cis regulatory variants that affect the expression of genes on the proto‐sex chromosomes, or trans effects of the proto‐Y chromosomes on genes elswhere in the genome. We used RNA‐seq to identify genes whose expression depends on proto‐Y chromosome genotype and temperature in adult male house flies. We found no evidence for ecologically meaningful temperature‐dependent expression differences of sex determining genes between male genotypes, but we were probably not sampling an appropriate developmental time‐point to identify such effects. In contrast, we identified many other genes whose expression depends on the interaction between proto‐Y chromosome genotype and temperature, including genes that encode proteins involved in reproduction, metabolism, lifespan, stress response, and immunity. Notably, genes with genotype‐by‐temperature interactions on expression were not enriched on the proto‐sex chromosomes. Moreover, there was no evidence that temperature‐dependent expression is driven by chromosome‐wide cis‐regulatory divergence between the proto‐Y and proto‐X alleles. Therefore, if temperature‐dependent gene expression is responsible for differences in phenotypes and fitness of proto‐Y genotypes across house fly populations, these effects are driven by a small number of temperature‐dependent alleles on the proto‐Y chromosomes that may have trans effects on the expression of genes on other chromosomes.

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Sex determination, the developmental process by which sexually dimorphic phenotypes are established, evolves fast. Species with polygenic sex determination, in which master regulatory genes are found on multiple different proto-sex chromosomes, are informative models to study the evolution of sex determination. House flies are such a model system, with male determining loci possible on all six chromosomes and a female-determiner on one of the chromosomes as well. The distributions of the two most common male-determining proto-Y chromosomes across natural populations suggests that temperature variation is an important selection pressure responsible for maintaining polygenic sex determination in this species. To test that hypothesis, we used RNA-seq to identify temperature-dependent effects of the proto-Y chromosomes on gene expression. We find no evidence for ecologically meaningful temperature-dependent expression of sex determining genes between male genotypes, but we identified hundreds of other genes whose expression depends on the interaction between proto-Y chromosome genotype and temperature. Notably, genes with genotype-by-temperature interactions on expression are not enriched on the proto-sex chromosomes. Moreover, there is no evidence that temperature-dependent expression is driven by chromosome-wide expression divergence between the proto-Y and proto-X alleles. Therefore, if temperature-dependent gene expression is responsible for differences in phenotypes and fitness of proto-Y genotypes across house fly populations, these effects are driven by a small number of temperature-dependent alleles on the proto-Y chromosomes.