Fig. 4.

Mrpex16 affects cuticle penetration, appressorium turgor generation, glycerol accumulation, lipid droplet degradation, and fungal pathogenicity in M. robertsii. (A) The penetration capacity of the WT and two ΔMrpex16 strains was assayed using cicada wings. Mycelial blocks inoculated on the cicada wings were incubated for 2 d (Left). Then, the cicada wings were removed, and the plates were incubated for an additional 5 d (Right). (B) Colony diameters of the WT and two ΔMrpex16 strains at 5 d after removing the cicada wings in the cuticle penetration assay. Data are shown as the mean ± SD of three biological replicates. Significant differences compared with those in WT were determined by Student's t test. **P < 0.01. The experiments were repeated twice with similar results. (C) Percentage of germinated conidia with appressoria relative to the total germinated conidia at 16 h and 24 h after induction in MM-Gly medium on hydrophobic plates. Data are shown as the mean ± SD of three biological replicates. The experiments were repeated twice with similar results. (D) Microscopic images of the WT and two ΔMrpex16 strains appressoria induced on cicada wings after immersion in PEG8000 for 10 min in an incipient cytorrhysis assay. (E) Percentage of appressoria induced on cicada wings that underwent incipient cytorrhysis after immersion in PEG8000 for 10 min. Data are shown as the mean ± SD of three biological replicates. Significant differences compared with those in WT were determined by Student's t test. **P < 0.01 and ***P < 0.001. (F) Intracellular glycerol levels in WT and ΔMrpex16 appressoria induced on hydrophobic plates. Data are shown as the mean ± SD of three biological replicates. Significant differences compared with those in WT were determined by Student's t test. *P < 0.05. The experiments were repeated twice with similar results. (G) Distribution of lipid droplets in appressoria of WT and ΔMrpex16 induced on cicada wings. Lipid droplets were stained with BODIPY. CO, conidium; AP, appressorium. (H) Survival of female adult A. stephensi mosquitoes following topical application of conidial suspensions (6 × 10⁶ conidia/mL) of the WT and two ΔMrpex16 strains. The control mosquitoes were treated with 0.01% Triton X-100. Each treatment was replicated three times, with 50 mosquitoes per replicate. Significant differences compared with those in WT were determined by the log-rank (Mantel–Cox) test. *P < 0.05 and ***P < 0.001. The experiments were repeated twice with similar results. (I) Survival of female adult A. stephensi mosquitoes after injection of conidial suspensions (138 nL of 1 × 10⁶ conidia/mL) of the WT and two ΔMrpex16 strains. The control mosquitoes were injected with 0.01% Triton X-100 in PBS. Fifty mosquitoes were used in each treatment. Significant differences compared with those in WT were determined by the log-rank (Mantel–Cox) test. ***P < 0.001. The experiments were repeated twice with similar results.