Temporal analysis of Plutella xylostella Masculinizer (PxyMasc) and doublesex (dsx) expression/splicing patterns in early eggs. Individual eggs were collected within 5 min of being laid and allowed to develop for 3, 6 and 24 h before being sampled (frozen in liquid nitrogen, RNA extracted, cDNA synthesized). Samples were used as template for paired PxyMasc/dsx PCR. A minimum of 25 eggs were obtained at each time point with the images above displaying representative samples from each group. Panel A shows results of dsx PCR, panel B shows results of PxyMasc PCR (PCRs in the same lanes in these two panels were run using the same sample, ie cDNA from the same egg), panel C shows controls run using sexed male (M) and female (F) pupal cDNA as well as a no template control (−). At 3 h postlaying, all eggs show female dsx splicing and no PxyMasc transcript is observable. At 6 h postlaying all individuals are showing both male (c. 200 bp) and female (c. 600 & 450 bp) dsx bands and all show full PxyMasc expression (c. 1200 bp) as well as a smaller PxyMasc transcript (c. 200 bp, Fig. 3B). At 24 h postlaying, samples showed either male dsx splicing (first three samples) or female dsx splicing (second three samples) with PxyMasc expression only remaining in those individuals showing male dsx bands.