qPCR for gene expression and data analysis

Levels of expression for genes of interest were quantified using the qPCR primers listed in Additional file 1: Table S1. Primers were designed using Primer3, and primer efficiency was verified using a 5 × 5 fold dilution series; primer efficiencies are reported in Additional file 1: Table S1. All qPCR reactions used the following thermal cycling conditions: 95 °C for 10 min, then 35 cycles of 95 °C for 15 s followed by 60 °C for 45 s, with the product verified by melt curve analysis, as well as Sanger sequencing (Macrogen USA) once for each primer set. We used Applied Biological Materials, EvaGreen 2X qPCR MasterMix according to manufacturers protocol, with a BioRad CFX96 qPCR thermal cycler.

For all immune challenges, we assayed the expression of each fly’s respective diptericin orthologue. In Serratia challenges, we also assayed attacin B (AttB) and drosocin. In Beauveria challenges, we also assayed a bomanin (Bom) gene (CG5791 in D. melanogaster and its respective orthologues in D. virilis and D. neotestacea), drosomycin in D. melanogaster, and drosocin in D. neotestacea and D. virilis.

For all qPCR reactions, target genes were run alongside a normalizing control gene (RpL28, RpL32, and RpL11 for D. neotestacea, D. melanogaster, and D. virilis respectively). Each reaction was run in triplicate, and replicates were considered consistent if the threshold cycle (C_T) of each replicate was contained within a 0.5 C_T boundary.

Gene expression analysis was performed using the 2^ΔΔCT method [48], and we report these data as boxplots using the ΔC_T values (ΔC_T = C_T target gene – C_T reference gene). Two-sample Welch’s T-tests of ΔC_T values were used to determine differences in expression profile in R 3.1 statistical software.