VRILLE shows high divergence among Higher Diptera flies but may retain role as transcriptional repressor of clock.

In the circadian system, the clock gene vrille (vri) is an essential component of the second feedback loop, being responsible in Drosophila for the rhythmicity of the Clock (Clk) gene transcription by its repression. Here we studied vri in a fruit fly pest, the Tephritidae Anastrepha fraterculus, aimingtoinvestigate its molecular evolution and expression patterns from whole-head extracts. We used a combination of transcriptomic, genomic and gene walking strategies to sequence and characterize Afravri in male and female head transcriptomes of A. fraterculus and detected two putative isoforms that may correspond to A and D vri isoforms of Drosophila. Both isoforms produced a full-length sequence that translates to 842 amino acids. While the protein sequence showed significant divergence to orthologous sequences from other organisms, the bZIP domain was highly conserved. Molecular evolutionary analyses showed that vri in higher Diptera flies has been evolving under positive selection. A more detailed analysis showed positive selection also in Tephritidae with 29 sites evolving under positive selection in comparison with Drosophilidae. Real time expression analysis in LD and DD conditions showed cyclic expression of Afravri mRNA with oscillation opposite to AfraClk, suggesting that VRI may also behave in Anastrepha as a transcriptional repressor of Clk, providing another indication that higher Diptera might share common interlocked transcript-translation feedback loops (TTFLs) mechanisms that differ from other insects in target genes.