Progression of Plasmodium berghei through Anopheles stephensi is density-dependent

Abstract

It is well documented that the density of Plasmodium in its vertebrate host modulates the physiological response induced; this in turn regulates parasite survival and transmission. It is less clear that parasite density in the mosquito regulates survival and transmission of this important pathogen. Numerous studies have described conversion rates of Plasmodium from one life stage to the next within the mosquito, yet few have considered that these rates might vary with parasite density. Here we establish infections with defined numbers of the rodent malaria parasite Plasmodium berghei to examine how parasite density at each stage of development (gametocytes; ookinetes; oocysts and sporozoites) influences development to the ensuing stage in Anopheles stephensi, and thus the delivery of infectious sporozoites to the vertebrate host. We show that every developmental transition exhibits strong density dependence, with numbers of the ensuing stages saturating at high density. We further show that when fed ookinetes at very low densities, oocyst development is facilitated by increasing ookinete number (i.e., the efficiency of ookinete-oocyst transformation follows a sigmoid relationship). We discuss how observations on this model system generate important hypotheses for the understanding of malaria biology, and how these might guide the rational analysis of interventions against the transmission of the malaria parasites of humans by their diverse vector species.

Figure 1. Changes in Parasite Abundance during Development within the Mosquito

Reported changes in numbers of P. berghei as it develops in An. stephensi, starting from an intake of 10⁴ macrogametocytes. Note the use of a log-scale on the y-axis. Figure adapted from Sinden [87], based on data from Alavi et al. [55].

Figure 2. Analysis of Parasite Densities in the Transition from Macrogametocytes to Ookinetes

(A) Frequency distribution of the number of ookinetes per mosquito. Observed frequency (red bars), expected frequency according to negative binomial distribution (blue bars) with k = 0.45 (an inverse measure of the degree of overdispersion; i.e., the lower the value of k, the greater the departure from the Poisson, random distribution), estimated by maximum likelihood. (B) Maximum likelihood estimates of parameter k (for each macrogametocyte density) against arithmetic mean ookinete density. The degree of overdispersion decreases (k increases) with mean ookinete density with a saturating relationship when the outlying red point (from OF's experiments) is excluded. (C) WM number of ookinetes per mosquito against number of macrogametocytes per bloodmeal. Ookinete density increases nonlinearly with macrogametocyte density when the outlying red point (from JM's experiment) is excluded. Error bars denote standard errors of WMs. (D) Number of ookinetes per individual mosquito against number of macrogametocytes per bloodmeal. The fitted curve corresponds to a saturating function (with underling nonlinear relationship between overdispersion parameter and mean ookinete density). All data pertain to P. berghei in An. stephensi. Markers in (B–D) refer to three experiments by OF (blue) and one by JM (green).

Figure 3. Analysis of Parasite Densities in the Transition from Ookinetes to Oocysts

(A) WM number of oocysts per mosquito against number of ookinetes per μl of blood. Mean oocyst density is related to ookinete density by a sigmoid function. Error bars denote standard errors of the WMs. (B) Number of oocysts per individual mosquito against number of ookinetes per μl of blood. The black line is fitted to the combined data; the brown line to data from [88]; the green line to data from JM; and the pink line to data from experiments by JW. In all cases the fitted curve corresponds to a sigmoid function (with an underlying nonlinear, power relationship between overdispersion and mean oocyst density). Assuming a bloodmeal volume of 2.13 μl, ookinete density can be expressed per mosquito by multiplying by 2.13. All data pertain to P. berghei in An. stephensi. Markers refer to one experiment by JM (green), three by JW (pink/violet), and five by [88] (orange/yellow/brown).

Figure 4. Analysis of Parasite Densities in the Transition from Oocysts to Sporozoites in the Salivary Glands

(A) WM number of sporozoites in the salivary glands against WM number of oocysts per mosquito. Mean sporozoite density increases linearly with oocyst density. Error bars denote standard errors of the WMs. (B) Number of salivary gland sporozoites per individual mosquito against the WM number of oocysts per mosquito. The fitted curve corresponds to a saturating function (with underling linear relationship between overdispersion and mean ookinete density). All data pertain to P. berghei in An. stephensi. Markers refer to three experiments by JW.

Figure 5. Relationship between the Number of Salivary Gland Sporozoites and Sporozoites Transferred in the Bite

Data from Medica and Sinnis [6] (P. yoelii in An. stephensi) illustrating the relationship between the number of sporozoites counted in dissected salivary glands and the number of sporozoites observed in the saliva ejected by a single bite. Note the use of a logarithmic scale on the x-axis.

Figure 6. Reduction in Oocyst Prevalence Induced by a 90% Reduction in Intensity at Different Initial Densities

A 90% blockade in intensity at varying oocyst numbers by a theoretical intervention results in the greatest reductions in prevalence of infected mosquitoes when mean oocyst numbers are low. Data from Medley et al. [18].