Global depletion of c3a.1 decreases neutrophil recruitment to, and subsequent survival of, localized bacterial infection. (A) RT-qPCR validation of c3a, c3b, and c5a orthologue expression in pooled WT and c3a.1−/− (sa31241) whole zebrafish larvae, normalized to WT expression for each gene and to ef1α, with data expressed as mean + /- SEM. Data comprise 3 independent experiments, performed in triplicate, n = 50 larvae per condition per experiment. (B) Experimental setup. c3a.1+/+ (n = 71, 1 hpw; 65, 6 hpw) and c3a.1−/− (n = 71, 1 hpw; 68, 6 hpw) larvae were inoculated with 1000 CFU Pseudomonas aeruginosa in the left otic vesicle and subsequently stained with Sudan Black B. The otic vesicle region (box) was imaged. (C) Representative images and (D) quantification of neutrophil recruitment following otic vesicle infection, with data expressed as mean + /- SEM. Each dot represents one larva; colors represent results of 3 independent experiments. ***p < 0.001 (E) c3a.1+/+ (n = 24) and c3a.1−/− (n = 32) larvae were infected with 7500 CFU Pseudomonas aeruginosa in the left otic vesicle and survival was tracked over 5 days post-infection. (F) To test whether survival was neutrophil-dependent, c3a.1+/+ (n = 16) and c3a.1−/− (n = 14) larvae with neutrophils that are mcherry-labeled and carry a mutation in rac2 rendering them migration-defective (Tg(mpx:rac2D57N-mcherry)) were infected with Pseudomonas aeruginosa as in C and survival was tracked over 5 days post-infection. E and F each comprise 3 independent experiments.
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