Anopheles mortality is both age- and Plasmodium-density dependent: implications for malaria transmission

Abstract

Background: Daily mortality is an important determinant of a vector's ability to transmit pathogens. Original simplifying assumptions in malaria transmission models presume vector mortality is independent of age, infection status and parasite load. Previous studies illustrate conflicting evidence as to the importance of Plasmodium-induced vector mortality, but very few studies to date have considered the effect of infection density on mosquito survival.

Methods: A series of three experiments were conducted, each consisting of four cages of 400-1,000 Anopheles stephensi mosquitoes fed on blood infected with different Plasmodium berghei ookinete densities per microlitre of blood. Twice daily the numbers of dead mosquitoes in each group were recorded, and on alternate days a sample of live mosquitoes from each group were dissected to determine parasite density in both midgut and salivary glands.

Results: Survival analyses indicate that mosquito mortality is both age- and infection intensity-dependent. Mosquitoes experienced an initially high, partly feeding-associated, mortality rate, which declined to a minimum before increasing with mosquito age and parasite intake. As a result, the life expectancy of a mosquito is shown to be dependent on both insect age and the density of Plasmodium infection.

Conclusion: These results contribute to understanding in greater detail the processes that influence sporogony in the mosquito, indicate the impact that parasite density could have on malaria transmission dynamics, and have implications for the design, development, and evaluation of transmission-blocking strategies.

Figure 1

Schematic representation of the experimental design. Three experiments were conducted each consisting of 4 cages of An. stephensi mosquitoes, represented by boxes in the figure.

Figure 2

Kaplan-Meier survival curves with time post-engorgement for each group of An. stephensi mosquitoes. (A) Experiment 1. Colors; black = 0 ookinetes per μl of blood fed; red = 100 ookinetes per μl of blood fed; green = 400 ookinetes per μl of blood fed; blue = 2,000 ookinetes per μl of blood fed. (B) Experiment 2. Colors as in panel A: (C) Experiment 3. Colors; black = 0 ookinetes per μl of blood fed; dark red = 50 ookinetes per μl of blood fed; dark green = 250 ookinetes per μl of blood fed, dark blue = 1,000 ookinetes per μl of blood fed.

Figure 3

Mortality rate with time post-engorgement. Relationship between the mortality rate of An. stephensi mosquitoes fed on blood containing different densities of P. berghei ookinetes and time post-engorgement (days). Markers correspond to the observed death rates plotted for the mid-point of each time interval. The lines are the best fit hazard model defined in Equation (2) for each parasite density, and the shaded area corresponds to 95% confidence intervals. Panels A to G represent increasing ookinete density per μl of blood fed, with colors as in Figure 2; (A) Control (0 ookinetes). (B) 50 ookinetes. (C) 100 ookinetes. (D) 250 ookinetes. (E) 400 ookinetes. (F) 1,000 ookinetes. (G) 2,000 ookinetes. (H) Hazard curves from each of the parasite densities on a single axis to facilitate comparison; colors as in panels A to G, in order from lowest to highest at time post engorgement = zero (where curves cross the y-axis), 0, 50, 100, 250, 400, 1000 and 2000 ookinetes per μl of blood fed. Figure 4 illustrates how the parameters of the mortality function vary with ookinete density fed to the mosquitoes.

Figure 4

Relationship between parameters of the mortality function and ookinete density fed. Linear functions (as illustrated in Equation (2)) are fitted to the relationship between the parameter values of the mortality function and parasite density fed to each group of mosquitoes (ookinetes per μl of blood). Shaded areas represent 95% confidence intervals. (A) Parameter ν, which predominantly represents the increase in mortality rate with time-post feeding; parameter values (and 95% confidence intervals), ν₀ = 1.18 × 10^-4 (4.65 × 10^-5, 1.40 × 10^-4)**, ν₁ = 6.43 × 10^-8 (4.29 × 10^-8, 2.60 × 10^-7)**. (B) Parameter δ, which predominantly represents the initial decline in mortality rate with time post-feeding; δ₁ = -3.27 × 10^-3 (-4.13 × 10^-3, -1.31 × 10^-3)**, δ₁ = -1.30 × 10^-6 (-6.77 × 10^-6, -1.24 × 10^-6)*. (C) Parameter θ, which represents the mortality rate at the time of feeding; θ₀ = 3.09 × 10^-2 (9.69 × 10^-3, 5.06 × 10^-2)**, θ₁ = 1.07 × 10^-5 (5.99 × 10^-6, 5.33 × 10^-5)*. Significant p-values (* represents a p-value < 0.05 and ** represents a p-value < 0.001) indicate that the best-fit mortality function includes each of the parameter values in Equation (2).

Figure 5

Mosquito life expectancy. Life expectancy of mosquitoes maintained in the laboratory, plotted against time post-engorgement and number of ookinetes per μl of blood fed to the mosquitoes. The life expectancy values are generated from Equation (4) with S(t, K) as defined in Equation (3).