2.4 Candidate gene analysis
For the immune gene analysis, we used the candidate protein set from a previous RNA-seq study (Yang et al., 2020), which contains 166 genes suggested to be important in immune response against parasitoid wasp attack in Drosophila (Supporting information Tables S1), subdivided into seven functional immune gene categories: recognition (N =17), signalling (N =35), effector (N =21), proteases (N =35), haematopoiesis (N =31), melanisation (N =18), and wound healing (N =9). We then used BLASTP with an E-value≤1×10−6 and a bitscore>60 to identify these 166 immune proteins in the G. calmariensisproteome. We further assessed the accuracy of BLASTP results by comparing the BLAST hits with our functional annotation. Finally, to achieve a more conservative assessment of selection dynamics on immune genes, we only investigated candidate immune genes that were among the previously identified 4,154 SCOs, as including other loci in the genome may be confounded with recent gene birth death dynamics and generate spurious results in molecular analyses.