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Chromobacterium Csp_P Reduces Malaria and Dengue Infection in Vector Mosquitoes and Has Entomopathogenic and In Vitro Anti-pathogen Activities

Figure 2

Csp_P exposure causes high mortality in adults and larvae.

Csp_P was experimentally introduced into the adult midgut via either a sugar meal (A–D) or blood meal (E, F), and mortality was observed over 5–8 days. To introduce Csp_P via sugar meal, adults were allowed to feed for 24 h on 1.5% sucrose containing Csp_P liquid culture at a final concentration of ∼108 CFU/ml for An. gambiae and ∼106 or 1010 CFU/ml for Ae. aegypti. Csp_P ingestion significantly decreased survival in sugar-fed aseptic (i.e. pre-treated with antibiotics) An. gambiae (A, p<0.0001) and Ae. aegypti (B, p<0.0001). Each experiment was replicated three times. Total sample sizes: (A)PBS = 149; (A)Csp_P = 146; (B)PBS = 70; (B)Csp_P = 70. Ingestion of Csp_P significantly decreased survival in sugar-fed septic (i.e. not treated with antibiotics) An. gambiae (C, p<0.0001). In septic Ae. aegypti, survival was significantly decreased after feeding on a 1010 CFU/ml sugar meal (D, p<0.0001) but not after feeding on a 106 CFU/ml sugar meal (D, p = 0.08). Experiments in C and D were replicated twice. Total sample sizes: (C)PBS = 95; (C)Csp_P = 124; (D)PBS = 185; (D)Csp10∧6 = 223; (D)Csp10∧10 = 226. To introduce Csp_P via blood meal, Csp_P liquid culture (∼108 CFU/ml) was mixed 1∶1 with human blood/serum and fed to septic An. gambiae (E) and Ae. aegypti (F) adults. Experiments were replicated three times with total sample sizes: (E)PBS = 59; (E)Csp_P = 51; (F)PBS = 37; (F)Csp_P = 62. The effects of Csp_P on larval mortality were also tested by placing 2- to 4-day-old An. gambiae (G) and Ae. aegypti (H) larvae in water containing Csp_P at a starting concentration of 106 CFU/ml and monitoring survival over 5 days. Experiments were replicated 2–3 times with final sample sizes: (G)PBS = 80; (G)Csp_P = 60; (H)PBS = 100; (H)Csp_P = 60. P values reported above were obtained by performing pairwise Log-Rank Tests between PBS and Csp_P treatments. Survival curves were fitted using the Kaplan-Meier method. Vertical tick-marks indicate censored samples; in C and D multiple individuals were dissected on each day to measure Csp_P prevalence and bacterial load for Figure 1.

Figure 2

doi: https://doi.org/10.1371/journal.ppat.1004398.g002