Figure 1. Effects of time-of-day of blood meal and diurnal temperature fluctuation on vector competence of A. gambiae mosquitoes infected with P. falciparum malaria.

Mosquitoes were offered infected blood meals at a different time-of-day (18:00h [ZT12], 00:00h [ZT18], or 06:00h [ZT0]) and kept under either constant (i.e. 27°C with a Diurnal Temperature Range [DTR] of 0°C) or fluctuating (i.e. 27°C with a DTR of 10°C) temperature regimes. There is no effect of time-of-day of blood feeding on vector competence (oocyst or sporozoite prevalence) under constant temperature conditions but a significant increase in competence for mosquitoes feeding in the evening (18:00h; ZT12) and a significant reduction in competence for those feeding in the morning (06:00h;ZT0), relative to those feeding at midnight (00:00h; ZT18) under realistic fluctuating temperatures. The scatter plots show oocyst intensity, with the data points representing the number of oocysts found in infected individual mosquitoes, and the horizontal lines the median. The pie charts show oocyst or sporozoite prevalence calculated as the proportion of infected mosquitoes revealed by dissection of midguts and salivary glands, respectively. Asterisks indicate statistically significant differences between treatments (** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant at P = 0.05; P-values were Bonferroni corrected after pairwise comparisons). n indicates the number of mosquitoes sampled from four replicate containers of mosquitoes from two biologically replicated infection experiments. Numbers in parentheses indicate Clopper-Pearson 95% confidence intervals. Forty mosquitoes were sampled daily from four replicate containers (10 per container) for dissecting midguts on 7-9 days post infection (dpi) or salivary glands on 14-16 dpi. Further details of the analysis are reported in Supplementary Table 3.