Fig 1.
A second blood meal increases oocyst size in a Lp-independent manner.
(A) dsGFP (Cntrl)- and dsLp-injected females were infected with P. falciparum 3 days post injection (inj) and then either provided a second uninfected blood meal 3 days post infectious blood meal (d pIBM) (two red circles) or maintained on sugar (one red circle). Infection outcomes in all groups were determined at 7–14 d pIBM (arrows). (B) Oocyst prevalence (P, pie charts) (χ2 test: χ2 = 4.3, d.f. = 3, n.s.) and intensity (Linear mixed model; FDR-corrected post-hoc Student’s t tests shown for all models) are not affected by a second blood meal at 3 d pIBM (#BF: n.s.), but intensity is lower in Lp-depleted groups (dsRNA: p<0.0001). (C) Oocyst size at 7 d pIBM is significantly increased in females fed twice in both injection groups (Linear mixed model; #BF: p<0.0001; dsRNA: n.s.). Horizontal bars indicate median values. n = numbers of mosquitoes analyzed from 3 different experiments. n.s. = not statistically significant. See S1–S4 Tables for details of statistical models.
Fig 2.
A second blood meal accelerates oocyst development.
(A–B) Immunofluorescence assay of oocysts from control females (A) fed once (one red circle) or (B) fed twice (two red circles) at 8 d pIBM. (C) Oocyst from a female fed twice showing the release of mature sporozoites at 10 d pIBM. Sporozoites are labelled with circumsporozoite protein CSP (magenta) and DNA is stained with DAPI (gray). In all panels, scale bar = 10 μm.
Fig 3.
Mosquitoes are infectious sooner following a second blood meal.
(A–B) Salivary glands of females fed twice (two red circles) show (A) a significantly higher prevalence (P, pie charts) of sporozoites at 8 and 10 d pIBM (χ2 test: 8 d, χ2 = 15, d.f. = 3, p = 0.0018; 10 d, χ2 = 43, d.f. = 3, p<0.0001; FDR-corrected post-hoc χ2 tests shown) and (B) significantly more sporozoites at 10 d pIBM (Linear mixed model; #BF: p = 0.0001; FDR-corrected post-hoc Student’s t tests shown) than females fed once (one red circle). Horizontal bars indicate median values. There is no difference in sporozoite prevalence at a later time point (χ2 test: 14 d, χ2 = 2.4, d.f. = 3, n.s.). (C) The EIP50 (time to 50% infectious–dotted line; values also shown ± 95% C.I.) of control (Cntrl) females fed a second time (blue solid line) is reduced by 2.3 d (21%) compared with controls fed once (orange solid line), as determined from the sporozoite prevalence data shown in (A) (z test, Z = 5.7, p<0.0001). Similarly, EIP50 is reduced by 2.4 d (20%) in Lp-depleted females fed a second time (purple dashed line) compared to those fed once (red dashed line) (z test, Z = 5.3, p<0.0001). Fitted logistic curves (lines), EIP50 ± 95% C.I. (shaded). n = numbers of mosquitoes analyzed from 4 different experiments. n.s. = not statistically significant. See S1–S4 Tables for details of statistical models.
Fig 4.
Relative R0 values across sub-Saharan Africa show increase in malaria transmission potential.
(A) Regions of sub-Saharan Africa with 1 to 12 months of average temperature in 27 ± 2°C. Our modeling analysis was restricted to locations and months of the year during which the average temperature was in this range, in order to align with our laboratory conditions. Areas in gray depict regions where there are 0 months with average temperature in that range, or where the predicted probability of An. gambiae complex is less than 5%. (B) The percent increase of using revised estimates of EIP50 (derived from two blood feeds) to R0 using the standard estimates of EIP50 (derived from a single blood feed). Models using a standard EIP parameter may underestimate R0 by an average of least 10.1%. In these regions, the average change during the months within the relevant temperature range is 10.5% (range: 10.1%–12.1%).
Fig 5.
Parasite developmental rates are further enhanced in eggless mosquitoes.
(A) Oocysts are significantly larger in Δzpg (eggless) mutant females at 7 d pIBM after both one or two blood meals (Linear mixed model; #BF: p = 0.0001; genotype: p<0.0001; FDR-corrected post-hoc Student’s t tests shown for all models) compared to controls (Cntrl). (B) Oocyst intensities are lower in Δzpg mutant females compared to controls (Linear mixed model; #BF: p = 0.041; genotype: p<0.0001), whereas oocyst prevalence (P, pie charts; χ2 test: χ2 = 6.5, d.f. = 3, n.s.) is unaffected. (C) Salivary glands sporozoite numbers are increased in both control and Δzpg mutant females at 10 d pIBM after a second blood meal (Linear mixed model; #BF: p<0.0001; genotype: p = 0.0056), with sporozoite prevalence (P, upper pie charts) also increased in the Δzpg mutant background (χ2 test, χ2 = 81, d.f. = 3, p<0.0001), significantly at the first blood meal. Pooling data shows higher sporozoite prevalence in Δzpg mutant population (P, lower pie charts) (Fisher’s exact test). Horizontal bars indicate median values. n = numbers of mosquitoes analyzed from 3 (A and B) or 4 (C) different experiments. n.s. = not statistically significant. See S1–S4 Tables for details of statistical models.