Extended Data Fig. 9: Night period dictates mosquitos efficient feeding behaviour and sporozoite increased motility.
From: Parasite and vector circadian clocks mediate efficient malaria transmission
A. Percent of mosquito biting across two times of the day (ZT4 and ZT16) on anesthetized mice or warm human blood was significantly higher at night (ZT16) compared to daytime (ZT4). Each point represents one replicate from 8 independent experiments, where 5-20 co-housed mosquitoes had access to a bloodmeal from one of two sources mentioned above. Percent values were derived from dividing the total number of fed mosquitoes that were co-housed by the total number of mosquitoes that were co-housed. Two-way ANOVA, adjusted for multiple comparisons ** p = 0.00257. There was no difference in percent of mosquito biting on different blood sources during the same time of day (Two-way ANOVA, p = 0.4622). Bars represent mean and error bars represent +/- SEM. B. Four independent normalized hemoglobin standard curves plotted from corresponding linear regression equation calculated from the absorbance (562 nm) values of diluted hemoglobin standards from four independent runs of the hemoglobin colorimetric assay. Individual points represent mean log2 absorbance of two technical replicates per independent run. Data are presented as mean values +/- SD. C. Quantification of hemoglobin in each mosquito midgut, as a proxy of blood ingested, after mosquito access to a bloodmeal for 30 min (n = 67 mosquito midguts for daytime group and n = 68 mosquito midguts for nighttime group; 4 independent experiments with 2 replicates per timepoint of 5-20 mosquitoes feeding on blood source; Mann Whitney test, *** p = 0.001, two-tailed). Hemoglobin concentrations (mg/mL) of all mosquitos from glytube feeding experiments, regardless of blood-fed status. Hemoglobin levels were quantified from linear regression equation generated from hemoglobin standard curves. Data are presented as mean values with 95% confidence intervals. D. Quantification of hemoglobin in each engorged mosquito midgut, as a proxy of blood ingested, after mosquito access to a bloodmeal for 30 min (n = 33 engorged midguts for daytime group; n = 50 engorged midguts for nighttime group; 4 independent experiments). Hemoglobin concentrations (mg/mL) of only blood-fed mosquitos from glytube feeding experiments. Quantified from linear regression equation generated from hemoglobin standard curves. Data are presented as mean values +/- SEM. E. Parasite load in livers of mice after bite of infected mosquitos at ZT4 or ZT16. Quantification of parasite load by qPCR analysis of Plasmodium berghei 18S expression in the livers of mice at 46 h post infection by bite of infected mosquitos for 25 min (n = 24 mice from 4 independent experiments; t test, ** p = 0.007). F. The percentage of sporozoites moving were quantified at each time of the day (ZT4 and ZT16) by taking the sum of sporozoites that moved in 1-5 trails, 6-10 trails, or > 10 trails category and dividing it by the total number of sporozoites imaged for each timepoint in 3 replicates (total 3 replicates) per timepoint. For ZT4, n = 143 sporozoites across all replicates. For ZT16, n = 128 sporozoites across all replicates. An average of 40 sporozoites were imaged per replicate per time of day. Statistical analyses were performed in prism using the student’s t-test. Data are presented as mean values +/- SD.
