Voriconazole is effective in chemically-immunosuppressed larvae. 2 dpf larvae were injected with A. fumigatus spores, immunosuppressive drugs and/or antifungal drugs and/or vehicle control were added to the larval water, and survival was tracked. Data represent at least three pooled replicates. P values and hazard ratios were calculated by Cox proportional hazard analysis. A. Experimental setup. B. Wild-type larvae infected with TBK1.1 were treated with 10 µM indomethacin or vehicle control and/or 1 µg/ml voriconazole or vehicle control. Average injection CFUs = 25. C. Wild-type larvae infected with TBK5.1 were treated with 10 µM dexamethasone or vehicle control and/or 1 µg/ml voriconazole or vehicle control. Average injection CFUs = 45. D. Neutrophil-defective mpx:rac2D57N larvae infected with TBK1.1 were treated with vehicle control (top), 10 µM indomethacin (bottom left), or 10 µM dexamethasone (bottom right) and 1 µg/ml voriconazole or vehicle control. Average injection CFUs = 43.

Azoles have differing efficacy and toxicity in dexamethasone-immunosuppressed larvae. 2 dpf wild-type larvae were injected with A. fumigatus spores (TBK1.1), 10 µM dexamethasone or vehicle control and antifungal drugs or vehicle control were added to the larval water, and survival was tracked. Data represent three pooled replicates. P values and hazard ratios were calculated by Cox proportional hazard analysis. A. Larvae were treated with vehicle control, 2.5 µg/ml posaconazole, 1 µg/ml itraconazole, 0.5 µg/ml isavuconazole, or 1 µg/ml voriconazole. Average injection CFUs = 39. B. Larvae were treated with vehicle control or 1 µg/ml itraconazole. Average injection CFUs = 70.

Azoles have differing efficacy in larvae lacking functional phagocytes. 2 dpf larvae were injected with A. fumigatus spores (TBK1.1), antifungal drugs or vehicle control were added to the larval water, and survival was tracked. Data represent three pooled replicates. P values and hazard ratios were calculated by Cox proportional hazard analysis. A. Neutrophil-defective mpx:rac2D57N or control mpx:rac2WT larvae were infected and treated with vehicle control, 2.5 µg/ml posaconazole, 1 µg/ml itraconazole, 0.5 µg/ml isavuconazole, or 1 µg/ml voriconazole. Average injection CFUs: mpx:rac2WT = 15, mpx:rac2D57N = 21. B. Wild-type or neutrophil-defective mpx:rac2D57N embryos were injected with Cas9 and gRNAs targeting irf8 or luciferase-targeting controls. After infection, larvae were treated with vehicle control, 2.5 µg/ml posaconazole, 0.5 µg/ml isavuconazole, or 1 µg/ml voriconazole. Average injection CFUs: WT+luc = 58, WT+irf8 = 117, mpx:rac2D57N+luc = 73, mpx:rac2D57N+irf8 = 80. C. Neutrophil-defective mpx:rac2D57N larvae homozygous for a stable irf8 loss of function mutation were infected and treated with vehicle control, 2.5 µg/ml posaconazole, 0.5 µg/ml isavuconazole, or 1 µg/ml voriconazole. Data from sibling larvae from this experiment are shown in Supp Fig S4. Average injection CFUs = 34.

Macrophages fight hyphae in control and posaconazole-treated larvae. Neutrophil-defective mpx:rac2D57N larvae with labeled macrophages (mpeg1:EGFP or mpeg1:H2B-EGFP) were infected with mCherry-expressing TBK5.1 A. fumigatus spores at 2 dpf and treated with vehicle control or 2.5 µg/ml posaconazole. Larvae were live-imaged every day for 5 dpi and macrophage number and fungal area were quantified. Data represent five pooled replicates. A. Example images showing macrophages surrounding hyphae. B. The number of macrophages at the infection site at 1 and 2 dpi is plotted. Each dot represents one larva, color-coded by replicate. Lines represent emmeans and error bars represent SEM. P values were calculated by ANOVA. C. For each larva that experienced germination and fungal growth, the maximum fungal area observed is plotted versus the number of macrophages at the infection site on the same day. Each dot represents one larva.

Posaconazole promotes survival of larval hosts by inhibiting fungal germination. Neutrophil-defective mpx:rac2D57N larvae with labeled macrophages (mpeg1:EGFP or mpeg1:H2B-EGFP) were infected with mCherry-expressing TBK5.1 A. fumigatus spores at 2 dpf and treated with vehicle control or 2.5 µg/ml posaconazole. Larvae were live-imaged every day for 5 dpi and macrophage numbers and fungal areawere quantified. Data represent five pooled replicates. A. Cumulative percentage of larvae that experience germination (solid line) or invasive hyphae (dotted line). P values and hazard ratios were calculated by Cox proportional hazard analysis. B. Of the larvae that experience germination by 3 dpi, the percentage that progress to invasive hyphae during the 5 day experimental period was calculated. P values were calculated by Fishers’ exact test. C. Of the larvae that experience germination by 4 dpi, the change in fungal area to the next day was calculated. Each dot represents one larva, color-coded by replicate. Lines represent emmeans and error bars represent SEM. P values were calculated by ANOVA. D, E. Example images of larvae that experience germination and control hyphal growth (D) or experience uncontrolled tissue-invasive hyphal growth (E). F. Heatmap representing the hyphal area measured from all larvae that experience germination at some point during the 5 days of infection. Each column represents one larva, imaged every day for 5 days or until the larva succumbed to infection.