Defining infection parameters. (A) Flow chart showing the workflow of pilot experiments. Hindbrain infections were performed at the prim-25 stage, and fish were then screened to ensure they received the correct inoculum (10–25 cells). At 4–6 hours postinfection, fish were imaged by confocal microscopy to score fungal phagocytosis; survival was monitored out to 72 hpi. (B) Example Kaplan-Meier survival curves pooled from three experiments showing the fish injected with PBS (control, n = 83), SN250 (WT, n = 61), adr1∆/∆ (n = 63), or mad2∆/∆ (n = 57). Fish injected with mad2∆/∆ showed increased survival compared to SN250 (P = 0.0001). (C) Survival of fish injected with each strain at 72 hpi in three independent experiments. Individual points represent biologically independent experiments on different days. Bars show means and standard deviations with SN250 in red to depict WT cutoff range for inclusion of experiments. Significant differences in survival curves were determined by Mantel-Cox log-rank tests comparing the mutant strain to SN250 from data pooled from three biological replicates of the same experiments. Two mutants were tested per experiment, and Bonferroni corrections were performed. (D) Representative images of hindbrain ventricle infection to score fungal phagocytosis at 4 hpi. C. albicans initial inoculum was stained with Calcofluor white, shown in blue. The hindbrain ventricle is outlined by white dashed lines. Scalebar is 50 µm. Arrows point to extracellular Candida, while arrowheads point to intracellular Candida. (E) Quantification of the percent of intracellular Candida. Fungal cells were scored as intracellular or extracellular from z-stack slices (using Calcofluor fluorescence for the fungi and differential interference contrast [DIC] for imaging the phagocytes) of individual fish taken at 4–6 hpi for each strain. Based on at least 19 fish from at least three independent experiments. Significance and effect size were determined as described in Materials and Methods and based on reference 38. *P < 0.05, **P < 0.01, ***P < 0.001.
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