Fig 1.
Mosquito infection by diverse fungal strains.
(A) Survival curves of fungal-challenged mosquitoes. Graph represents 3 independent experiments and data was analyzed with Log-rank Test (GraphPad Prism 7) (B) Entomopathogenic fungi infection stages: conidia (top), hemocytes and blastospores (middle), and hyphal growth on mosquito cadavers (bottom). Black arrows indicate hemocytes while red arrows indicate blastospores. No blastospores were found in T. roseum-challenged mosquitoes.
Fig 2.
Recognition of diverse fungal strains by the mosquito immune system.
Relative expression of fungal recognition genes CLSP2 and TEP22 in the midgut and fat body at 3d and 6d PI. Data represents the fold change in expression from three independent experiments. Data was analyzed by one-way ANOVA with Dunnett’s post-test; * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.
Fig 3.
Elicitation of innate immune signaling pathways following fungal infection is time, tissue and fungal strain-specific.
(A) Gene expression analysis of REL1 (Toll pathway), REL2 (IMD pathway), STAT (JAK-STAT pathway) and JNK (JNK pathway) in the midgut and fat body at 3d and 6d PI. Data represents the fold change in expression from at least three independent experiments. (B) Heatmap generated from the antimicrobial peptide gene expression following fungal infection in the midgut and fat body at 3d and 6d PI. Heat-map from qPCR data represents the median of log2 fold change values from three independent experiments with red representing higher expression levels and green lower expression levels compared to the control. CECG, cecropin G; DEFC, defensin C; ATTA, attacin; LYSC, lysozyme C. Data was analyzed by one-way ANOVA with Dunnett’s post-test; * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.
Fig 4.
Entomopathogenic fungal infection results in the downregulation of PPO gene expression and reduction of phenoloxidase activity.
Gene expression profiles of (A) PPO3 and (B) PPO5 in the midgut and fat body at 3d and 6d PI. Data represents the fold change in expression from three independent experiments. Data was analyzed by one-way ANOVA with Dunnett’s post-test. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Phenoloxidase activity (Vmax) was evaluated from whole-body macerates of mosquitoes at (C) 3d and (D) 6d PI. Data represents samples from 3 independent experiments. Data was analyzed via ANOVA-Kruskal-Wallis followed by Dunn’s multiple comparison test. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.
Fig 5.
Entomopathogenic fungal infection leads to dysregulation of the mosquito midgut homeostasis.
Relative quantification of (A) bacterial 16s rRNA in the mosquito midgut at 3d and 6d PI and (B) bacterial 16s rDNA at 6d PI. ** P<0.01. (C) Relative bacterial OTU abundance at the genus level from mosquito midguts collected at 6d PI. Labels on the x-axis represent the replicate groups under each treatment. (D) Principal coordinate analysis (PCoA) depicting patterns of beta diversity for the mosquito midgut bacterial communities under different fungal infections. PCoA was based on the Bray-Curtis dissimilarity matrix.
Fig 6.
Dysbiosis of the mosquito gut occurs via modulation of gut-homoeostasis-related genes and reduction of ROS activity in the midgut.
Expression profiles of (A) MESH and (B) DUOX1 in the mosquito midgut at 3d and 6d PI. Data represents the fold change in expression from at least three independent experiments. (C) Hydrogen peroxide release (ROS) from the mosquito midgut at 6d PI. (D) Midgut ROS activity was imaged using DHE at 6d PI. Top panels show DHE fluorescence (red) while lower panels show DAPI staining of nuclei (blue). Heat map depicting the differential expression of oxidant and antioxidant genes in the (E) midgut and (F) fat body at 3d and 6d PI. Data represents the fold change in expression from three independent experiments. Data was analyzed by one-way ANOVA with Dunnett’s post-test. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.