Quantifying Sexual Dimorphism in Larval Drosophila

We used a molecular method. βtubulin85D (FBgn0003889) is reported to be expressed specifically in testes [73 (link)] predicting a dimorphic pattern of βtub85D expression, high in males and low in female larvae. We first verified this by sexing a subset of larvae by eye on the basis of the larger testes versus ovary imaginal discs. We then quantified the level of βtub85D expression in the same larvae, normalized to reference genes elF1A (FBgn0026250) and αTub84B (FBgn0003884) (electronic supplementary material, table S2). As expected, larvae sexed as males had high βtub85D expression and larvae sexed as females had no βtub85D expression above background noise. Thus we used βtub85D expression assays to identify the sex of experimental larvae.