Silencing of adenosine kinase and adenylate kinase in hemocytes affects the SAM transmethylation pathway and immune response
(A) Total levels of AMP in hemocytes shown as the mean metabolite amounts expressed by the normalized peak area. Infection significantly increases the AMP level in control hemocytes (uninfected - Uninf - light gray vs. infected - INF - dark gray). Hemocyte-specific adenosine kinase Adk3 knockdown (Adk3 RNAi; uninfected - Uninf - light blue and infected - INF - dark blue) significantly decreases AMP during infection compared to control. (B) Methylation index, calculated as the ratio of SAM:SAH levels (peak areas; numerical values in S1_Data and S2_data), in control (uninfected - Uninf - light gray and infected - INF - dark gray) and in hemocyte-specific Adk3-RNAi and adenylate kinase Ak1-RNAi (uninfected - Uninf - light blue and infected - INF - dark blue). (C) Pupation is delayed (10 h) upon infection in control larvae (n = 270, uninfected dashed gray line and n = 240, infected solid black line) but significantly less (7 h) in hemocyte-specific Adk3 RNAi larvae (n = 275, uninfected dashed blue line and n = 275, infected solid blue line). (D) The number of lamellocytes is significantly lower in infected Adk3-RNAi (blue) larvae compared to infected control (gray). (E) Percentage of infected larvae surviving to adulthood is significantly lower in Adk3-RNAi (blue) compared to control (gray). (F) Pupation is delayed (9 h) upon infection in control larvae (n = 225, uninfected dashed gray line and n = 270, infected solid black line) but significantly less (6 h) in hemocyte-specific Ak1 RNAi larvae (n = 195, uninfected dashed blue line and n = 225, infected solid blue line). (G) The number of lamellocytes is significantly lower in infected Ak1-RNAi (blue) larvae compared to infected control (gray). (H) Percentage of infected larvae surviving to adulthood is significantly lower in Ak1-RNAi (blue) compared to control (gray). (A,B,D,E,G,H) Bars/lines represent mean values with 95% CI of uninfected (Uninf, light grey) and infected (INF, dark grey) control and uninfected (Uninf, light blue) and infected (INF, dark blue) Adk3-RNAi or Ak1-RNAi samples; each dot represents one biological replicate (numerical values in S1_Data and S2_data); asterisks represent significant differences between samples tested by unpaired t-test or ordinary one-way ANOVA Tukey’s multiple comparison test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). (C,F) Lines represent percentages of pupae at hours after egg laying (h AEL); rates were compared using Log-rank survival analysis.