Macrophages control spore germination and extracellular hyphal growth through Rac2 function. rac2+/–; mpx::mCherry-2A-rac2D57N adult fish were crossed to each other, and the resulting larvae were injected with YFP-expressing TBK1.1 (Af293) spores. Larvae were live imaged via confocal microscopy at 1–5 dpi and genotyped at the conclusion of the experiment. All larvae in the experiment carry the mpx::rac2D57N transgene. Data represent 3 pooled replicates of 22–31 larvae per replicate. (A and B) The cumulative percentage of larvae experiencing germination (A) and invasive hyphae (B) was calculated. P values and hazard ratios were calculated by Cox proportional hazard analysis. (C) Of larvae experiencing germination, the percentage that progress to invasive hyphae was calculated. P values calculated by Fisher’s exact test. (D) Of larvae that experience germination and invasive hyphae, the number of days between the observance of these forms of growth was calculated. Each symbol represents one larva, color coded by experiment; lines represent emmeans ± SEM. P values calculated by ANOVA. (E) Example images showing germination in larvae at 2 dpi and subsequent control of fungal growth (top) or invasive hyphae development and death (bottom). (F) In larvae that experience germination, the fungal (YFP+) area was measured at both 0 and 1 dpg. Each symbol represents one larva, color coded by experiment; lines represent emmeans ± SEM. P values calculated by ANOVA. (G) Survival of larvae was monitored for the 5 days of imaging. P values and hazard ratios calculated by Cox proportional hazard analysis.
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