Quantitative dynamics of Plasmodium yoelii sporozoite transmission by infected anopheline mosquitoes

Abstract

Malaria transmission begins with the injection of Plasmodium sporozoites into the skin of a vertebrate host by infected anopheline mosquitoes. Although the size of the sporozoite inoculum likely affects the course of the disease, the number of sporozoites injected by infected mosquitoes has not been determined in vivo. Using a quantitative PCR assay, we determined the number of sporozoites injected into mice by single mosquitoes. Analysis of 59 mosquito feedings showed that a single infected mosquito injected between 0 and 1,297 sporozoites, with a mean of 123 and a median of 18. Twenty-two percent of infected mosquitoes injected no sporozoites. The number of sporozoites injected was only weakly correlated to the salivary gland load. To better understand the large variability in sporozoite injection among mosquitoes, we quantified the sporozoites injected by individual mosquitoes on three different days. Approximately 20% of moderately to heavily infected mosquitoes injected few to no sporozoites on all 3 days, suggesting that some mosquitoes are poor transmitters of sporozoites. Other mosquitoes injected high numbers of sporozoites on at least one of the days observed and minimal numbers on the other day(s), supporting the hypothesis that sporozoite injection is discontinuous, a pattern that may aid in the establishment of malaria infection.

FIG. 1.

Mice in which the injection site is removed do not develop malaria infection. Five to eight infected mosquitoes were allowed to probe on a mouse's ear, and then the ear was either left intact or removed. Forty hours later, malaria infection in the livers of these mice was determined by RT-real-time PCR. Infection was quantified using a standard curve made from results for the livers of mice injected with known numbers of sporozoites. There were three mice per group, and the means with standard deviations are shown. The asterisk indicates that there was no detectable signal in this group.

FIG. 2.

Quantification of P. yoelii sporozoites in mouse skin. RNA was extracted from mouse ear homogenates containing the indicated number of sporozoites, and RT followed by real-time PCR was performed using parasite-specific primers. Shown is the PCR cycle number at which each sample became positive over background fluorescence.

FIG. 3.

Quantification of sporozoite injection by individual mosquitoes. (A) Shown is the relationship between sporozoite injection and salivary gland load. Individual mosquitoes were allowed to probe for 3 min on the ear of an anesthetized mouse. The ear of the mouse and the salivary glands of the mosquito were removed, and the number of sporozoites in each was quantified by RT-real-time PCR. The regression line for the data is shown. (B) The distribution frequency of the data shown in panel A is plotted to show the percentage of mosquitoes that injected a given range of sporozoites. (C) For each of the mosquitoes shown in panel A, the percentage of the salivary gland load that was injected during probing (transmission efficiency) was determined, and shown is the percentage of mosquitoes which corresponds to each of these values.

FIG.4.

The ejection pattern of individual mosquitoes over time. Individual mosquitoes were allowed to feed on the ears of anesthetized mice for either three consecutive days (days 15, 16, and 17 p.i.) (A) or three alternate days (days 15, 17, and 19 p.i.) (B). After each feeding, the number of sporozoites injected into the ear was determined by RT-real-time PCR. After the last feed, the salivary gland load of the mosquito was determined by visual inspection and in most cases also by RT-real-time PCR. Shown for each mosquito are the numbers of sporozoites injected during each of three feedings plotted against the salivary gland load/100 of that mosquito. The designations light, moderate, and heavy indicate the level of gland infection as determined by microscopy. Mosquitoes for which only visual assessment of gland infection was available are indicated with an asterisk. Zeros denote no detectable sporozoites in the skin on all three days.