Fig 1. Glyphosate inhibits G. mellonella melanization and increases infection susceptibility.

(A) Glyphosate inhibits the phenoloxidase activity of 1:10 dilutions of hemolymph without exogenously added L-DOPA. (B) AMPA, a primary metabolite of glyphosate, inhibits G. mellonella phenoloxidase-mediated melanization similar to glyphosate. Error bars in (A, B) represent ± SD (C) G. mellonella larvae drugged with glyphosate solution (10 μl of 1 mM) in PBS and infected 5 hours posttreatment with 10⁴ cells of WT C. neoformans die rapidly compared to PBS-treated controls. Death events were recorded daily. Each infection condition represents survival of 95 animals, pooled together from 4 biological replicates, and 6 total technical replicates. Statistical significance was assessed by Gehan–Breslow–Wilcoxon test, which we used to place weight on early time points in the survival curve. We used this test because we expected to see the glyphosate-mediated differences early in the infection due to the timing of the glyphosate treatment. Since where the expected effects of the one-time pretreatment with glyphosate would be. (D) The size of the dark melanized particles within nodules upon C. neoformans infection are significantly smaller in the glyphosate-treated (10 μl of 1 mM) infected groups compared to the PBS-treated infected groups, which were analyzed for significance using a nested nonparametric Mann–Whitney–Wilcoxon rank test. Horizontal bar represents the median value and the error bars represent the 95% confidence interval (E) The degree of melanin encapsulation of the yeast within the nodule is also reduced in the glyphosate-treated (10 μl of 1 mM) groups, as measured on a scale of 0 (no melanin encapsulation) to 4 (very high levels of melanin encapsulation) as demonstrated in (F). Numbers in each bar represent the number of encapsulated C. neoformans for each score. Statistical significance was assessed using a chi-squared table test. Data in (D) and (E) represent data over 3 independent replicates with 3 larvae used per condition per replicate. (G) Representative brightfield micrographs showing the hemocyte and nodule formation in the different treatment groups at 20× and 100× magnification. Scale bars represent 10 μm. Nested nonparametric Mann–Whitney–Wilcoxon rank test performed using R for R 4.0.2 GUI 1.72 for Mac OS at https://www.r-project.org/ (R Core Team, 2020) and the nestedRanksTest package (Version 0.2, D.G. Scofield, 2014) [77]. All other statistical analyses performed using GraphPad Prism version 8.4.3 for Mac OS, GraphPad Software, San Diego, California, USA, www.graphpad.com. For underlying data, please see Data Availability section and/or S1 Table. See also S1 Fig. AMPA, aminomethylphosphonic acid; L-DOPA, 3,4-dihydroxyphenylalanine; PO, phenoloxidase; WT, wild-type.