Fig. 4

Effect of mating and infection on protein synthesis. A Representative image of puromycin incorporation in nascent polypeptides of the fat body tissue detected using anti-puromycin antibody and Western Blotting. Secondary antibodies labeled with different fluorophores detected puromycin (Green, 800nm) and Actin (Red, 700 nm). The fat bodies from Mated-Infected (MI) produce noticeably less protein than those of the other treatments. Virgin-Uninfected (VU), Virgin-Infected (VI), Mated-Uninfected (MU), and Mated-Infected (MI) represent the four treatments. Negative control (NC) (A and D) shows proteins from fat body tissues which were not incubated with puromycin. B Quantification of relative protein synthesis using puromycin incorporation from four treatments (VU, VI, MU, and MI) (n = 5, 10 flies per treatment). Treatments not connected by the same letter are significantly different (Tukey’s HSD, p < 0.05). Virgin-Infected females synthesize significantly more protein than Mated-Infected females. C Cox proportional hazard analysis showing rescued post-infection survival (p < 0.0001) of cycloheximide (CHX) pre-treated mated females (CHX- Mated-Infected) compared to non-treated Mated-Infected (n = 35–40 flies per treatment per replicate, three independent replicates). D Representative Western blot image of puromycin incorporation from CHX pre-treatment of mated females (CHX). CHX treatment partially rescues protein synthesis in response to infection compared to non-treated mated females (MI) (paired Student’s t-test, p < 0.05). Puromycin incorporation was measured after 6 h of infection. E The number of unique immune peptides detected per sample in hemolymph samples collected from VI, MI, and CHX females (n = 7, 3 flies per sample). Fewer immune peptides were detected in MI compared to VI (paired Student’s t-test, p < 0.01) and CHX (paired Student’s t-test, p < 0.05). (F) Heatmap showing the proportion of samples per treatment in which each peptide was detected. Darker colors in the heatmap indicate a higher proportion of samples in which a given peptide was detected. Significance levels: ns not significant, (*) p<0.05, (**) p<0.01, (***) p<0.001