Fig. 4
Persistence of adulticidal activity in cell-free preparations of C.sp_P. a C.sp_P adulticidal activity is not retained after removal of live cells from planktonic culture. C.sp_P was grown at 30 °C with shaking for approximately 16 or 72 h and diluted to 1.0 (± 0.1) OD600 with additional sterile LB. An aliquot of each culture was then filtered through a 0.2 μm filter to remove live bacterial cells. Each culture preparation or LB as a control was mixed 1:1 with 3% sucrose (1.5% sucrose final concentration) and provided to An. gambiae adult females for 24 h. Experimental sugar meals were then removed, and all treatments were provided 10% sucrose ad libitum. Survival was monitored for seven days. The experiment was repeated three independent times, with 17–25 individuals per treatment per replicate. Survival of each treatment was compared to the LB control using a Cox proportional hazards model: LB vs C.sp_P 16hr Plank Unfilt, z = 7.11, P < 0.0001; LB vs C.sp_P 16hr Plank Filt, z = -1.76, P = 0.078; LB vs C.sp_P 72hr Plank Unfilt, z = 9.57, P < 0.0001; LB vs C.sp_P 72hr Plank Filt, z = -3.25, P = 0.001. b C.sp_P adulticidal activity is retained after removal of live cells from biofilm culture media. C.sp_P was grown in biofilm conditions for 72 h at which point the biofilm and the LB media overlaying the biofilm (i.e. biofilm supernatant) were collected. Both biofilm and supernatant were filtered through a 0.2 μm filter to remove live bacterial cells. Each culture preparation, LB or PBS were mixed 1:1 with 3% sucrose, provided to An. gambiae adult females for 24 h and mortality was monitored for 7 days. Control for all samples is PBS with the exception of C.sp_P biofilm supernatant, for which the control is LB. All treatments caused a significant reduction in survival except filtered C.sp_P biofilm: PBS vs C.sp_P Planktonic, z = 6.13, P < 0.0001; PBS vs C.sp_P Biofilm, z = 11.34, P < 0.0001; PBS vs C.sp_P Biofilm Filtered, z = -0.33, P = 0.745; LB vs C.sp_P Biofilm Sup. Filtered, z = 6.66, P < 0.0001. The entire experiment was repeated four independent times with 18–40 individuals per treatment per replicate, and the data were analyzed using a Cox proportional hazards model