Fig. 4

Growth and rosette formation of LpAsp and LpCht mutants, as well as their infection in honey bees. a Growth rates of WT (circle) and homozygous (-/-) mutant strains for LpAsp (triangle denotes clone A9; square denotes clone B6), and LpCht (reversed triangle denotes clone D11; diamond denotes clone E6) in modified FP-FB medium were monitored at 28 °C over 4 days (n = 3). Symbols represent mean values ± standard deviation (SD) (error bars). b Microscopic images of parasites in the medium captured 5 days after culture initiation, showing rosettes of various sizes. c Count of rosettes within three different areas with the individual parasites (n = 3). Symbols represent mean values ± SD (error bars); statistical analysis was performed using the Dunnet test (one-tailed). Asterisks indicate statistical significance at *** P < 1.9E−06. d, e The relative abundance of L. passim in individual honey bees (n = 24) at 14 days post-infection was compared between WT and homozygous mutants (-/-) of LpAsp (d; clones A9 and B6) or LpCht (e; clones D11 and E6). One sample infected by the WT parasite was set at the reference value of 1, and symbols represent the median with 95% confidence interval. Statistical analysis was performed using the Kruskal–Wallis test followed by the Steel test. LpAsp, L. passim aspartyl protease; LpCht, L. passim chitinase; ns, not significant; WT, wild type