Direct microscopic quantification of dynamics of Plasmodium berghei sporozoite transmission from mosquitoes to mice

Abstract

The number of malaria sporozoites delivered to a host by mosquitoes is thought to have a significant influence on the subsequent course of the infection in the mammalian host. We did studies with Anopheles stephensi mosquitoes with salivary gland infections of Plasmodium berghei sporozoites expressing a red fluorescent protein. After individual mosquitoes fed on an ear pinna or the ventral abdomen of a mouse, fluorescence microscopy was used to count numbers of sporozoites. Mosquitoes allowed to feed on the ear for periods of 3 versus 15 min deposited means of 281 versus 452 sporozoites, respectively, into the skin; this may have epidemiological implications because mosquitoes can feed for longer periods of time on sleeping hosts. Mosquitoes feeding on the ventral abdomen injected sporozoites not only into the skin but also into the underlying peritoneal musculature. Although mosquitoes injected fewer sporozoites into the abdominal tissues, more of these were reingested into the mosquito midgut, probably a consequence of easier access to blood intake from the abdominal area. The most consistent parameter of sporozoite transmission dynamics under all conditions of mosquito probing and feeding was the relatively slow release rate of sporozoites (approximately 1 to 2.5 per second) from the mosquito proboscis. The numbers of sporozoites introduced into the host by mosquitoes and the transmission efficiencies of sporozoite delivery are multifactorial phenomena that vary with length of probing time, skin site being fed upon, and numbers of sporozoites within the salivary glands.

FIG. 1.

Micrographs of fluorescent P. berghei sporozoites in mouse ear after 3-min feeding by infected A. stephensi mosquito. (A) Full-skin-thickness biopsy specimen. (B) Isolated dermal layer. (C) Isolated epidermal layer. Panels A to C are at the same magnification. (D) Low-power view showing hematomas (Hem) and clumps of deposited sporozoites (Spz).

FIG. 2.

(A) Frequency distributions of numbers of sporozoites injected into pinna of mouse ear by mosquitoes. Percentages of mosquito population injecting defined numbers of sporozoites within 3-min versus 15-min periods of feeding. (B) Frequency distributions of transmission efficiency into ear pinna by mosquitoes within 3-min versus 15-min periods of feeding. The transmission efficiency equals the number of sporozoites found in tissue divided by the number calculated to have originally been in the salivary glands.

FIG. 3.

Micrographs of fluorescent P. berghei sporozoites in mouse after 3-min feeding on ventral abdomen by infected A. stephensi mosquito. (A) Bright-field image of peritoneal musculature after removal of overlying abdominal skin. Hematomas (Hem) are seen after mosquito feeding on skin. (B) Fluorescence imaging of same specimen at higher magnification. The micrograph is from the area outlined by the square in panel A. Scattered fluorescent sporozoites are in the upper-right quadrant of the micrograph. Red linear streaks adjacent to sporozoites are nonspecific fluorescence associated with penetration tracks from the probing proboscis. (C) Full-skin-thickness biopsy specimen. (D) Full-thickness peritoneal musculature biopsy specimen. Panels C and D are at the same magnification.

FIG. 4.

(A) Frequency distributions of numbers of sporozoites injected into abdominal skin and subcutaneous tissue by mosquitoes. Percentages of mosquito population injecting defined numbers of sporozoites within 3-min versus 15-min periods of feeding. (B) Frequency distributions of transmission efficiency into abdominal skin and subcutaneous tissue by mosquitoes within 3-min versus 15-min periods of feeding. Transmission efficiency is defined in the legend for Fig. 2.

FIG. 5.

Micrographs of ear pinna (A) versus abdominal skin (B) after intravenous injection of fluorescein-labeled dextran to compare degrees of vascularization of tissues. Fluorescence microscope imaging. Both micrographs are at the same magnification.