Sex-specific expression during embryonic development of Anopheles gambiae

Currently employed vector control strategies are experiencing resistance that threaten to limit their effectiveness in controlling malaria transmission. Therefore, development of new genetic vector control strategies may benefit efforts to curb the devastating effects of malaria. Sex-specific gene expression has not been studied in mosquito embryos thus far, but its understanding may lead to creation of new tools for vector control. Here we analysed sex-specific transcription using firstly a targeted gene approach and secondly de novo transcriptional profiling of male and female embryonic transcriptomes. Two genes, doublesex (dsx) and fruitless (fru), belonging to sex determination/differentiation pathway, have sex-specific transcripts described previously in adult An. gambiae, and here, we present evidence that both dsx and fru are also sex-specifically spliced in the embryonic stages. dsx, a final gene in the sex determination pathway and a key regulator of sexual differentiation, is maternally deposited as a female transcript, but establishes a persistent pattern of alternative sex-specific splicing eight hours after egg laying (AEL). fru, a modulator of adult male behaviour, has sex-specific transcription starting at least 16 h AEL. The An. gambiae sex determination pathway was further investigated through isolation of a putative homologue of the D. melanogaster sex determination gene transformer2 (tra2), which was knocked down via RNAi in an attempt to confirm its involvement in Anopheles sexual development. Using a transient reduction in transcript levels, an effect on sex determination phenotype was not observed. In addition, bioinformatics and degenerate PCR approaches identified a large number of candidates with sequence similarity to transformer (tra), though these candidates require additional characterisation. Previous work on sex-specific expression has taken an a priori approach, using microarrays for gene expression analysis. Here, a de novo approach was undertaken and an approximate ~500,000 transcript fragments have been independently sampled using the 454 platform from each transcriptome of male and female embryos and assembled into 17,492 and 16,899 contigs from male and female reads, respectively. In silico subtraction of the two RNA-seq databases revealed a large number of putative male or female specific transcripts, of which 60 were tested using RT-PCR. Among those, two transcripts, displaying male-specific expression, represented two novel genes (YOA and YOB) located on the Y chromosome and sharing partially overlapping exons. YOA is expressed from late larvae through to adulthood. YOB is expressed from 4 hr AEL and continues across all stages through to adulthood. Their amino acid translation has no significant similarity to known proteins, or conserved domains. Furthermore, a functional role could not be assigned since gene silencing approaches did not reveal an obvious phenotype. This study has also emphasised that the current AgamP3.6 gene build is not complete. Deep sequencing of the embryonic transcriptome has produced a large dataset containing 6,436 transcripts that map to previously unannotated genomic regions. These sequences likely represent extensions to untranslated regions, novel exons or unique gene sequences. Our study demonstrates that RNA-seq is a powerful tool to identify sex-specific expression in mosquito embryos and that sex-specific transcription starts very early during embryonic development. The remaining question is whether the data produced within this study can be exploited in the context of a genetic vector control strategy