Fig 6. Effects of blocking adenosine transport in immune cells by ENT2 RNAi.

(A) Srp>ENT2-RNAi reduces lamellocyte number compared to w larvae, and high dietary glucose (12%-glu) significantly increases the lamellocyte number in Srp>ENT2-RNAi. Each dot represents lamellocyte count per larva; lines indicate mean. Differences were tested by unpaired t test. (B) ENT2 mRNA expression. Comparison of ENT2 mRNA expression in various tissues in w larvae shows a strong expression in brain and lymph gland, increasing in both upon infection. Srp>ENT2-RNAi reduces the ENT2 expression below 20% both in the lymph gland and hemocytes. Values are mean ± SEM of relative expression (normalized to Rp49 mRNA) of three experiments; tested by two-way ANOVA. (C) Nutrient contents in the hemolymph and whole larval lysates. Circulating glucose does not increase in Srp>ENT2-RNAi upon infection. Circulating trehalose in Srp>ENT2-RNAi does not form the 6 hpi peak of w; arrows indicate no change (ns) in levels of infected Srp>ENT2-RNAi when compared between time points. Tissue trehalose shows similar pattern to w. Glycogen does not differ between control and infected Srp>ENT2-RNAi indicating an accumulation of stores even upon infection. Asterisks show statistical significance when compared between infected and control animals at indicated time points (black for w, green for Srp>ENT2-RNAi). Values are mean ± SEM of four experiments; tested by two-way ANOVA. For statistical analysis, see S2 Fig. (D) Incorporation of ¹⁴C-glucose into lipids and proteins is reduced upon infection in w larvae but significantly less so in Srp>ENT2-RNAi larvae. Arrows indicate infection-induced changes. For statistical analysis, see S14 Fig. (E) Srp>ENT2-RNAi significantly reduces the host resistance to parasitoid wasp as assessed from frequency of melanized eggs (9% versus 42%; n = 100 Drosophila larvae per genotype in five experiments) and emerged adult flies (7% versus 38%; n = 316 for w, 343 for Srp>ENT2-RNAi in three experiments). Values are mean ± SEM; tested by unpaired t test. (F) Uninfected Srp>ENT2-RNAi larvae (n = 377) pupate 8 h earlier than uninfected w larvae, and infection only delays their pupation by 2 h (n = 343). Compared using Log-rank survival analysis (p < 0.0001 for all comparisons). (G) Total amount of TAG and phospholipids in the fat body of w, adoR, and Srp>ENT2-RNAi larvae. While infection suppresses TAG storage in w and adoR, TAG grows unaffected by infection in Srp>ENT2-RNAi. Data are mean values of mass spectra peak area per sample ± SEM; tested by two-way ANOVA.