Malarial parasites complete sporogony in axenic mosquitoes

Summary To obtain sporogonic stages of malaria free from microbial contaminants for in vitro studies,Anopheles stephensi were reared under sterile conditions using a mosquito cell line as larval food. The adult females, kept in sterile humidified containers and allowed to engorge on parasitemic hamsters, supported the sporogonic development of the rodent malarial parasitePlasmodium berghei. In 10 experiments, the proportion of infected mosquitoes varied from 0 to 92%, and the geometric mean number of oocysts per female mosquito from 2.5 to 58,6, with a range of 1 to 548. The average number of salivary gland sporozoites per infected mosquito was determined by direct sporozoite counts in the pooled homogenate of the thoraces of all female mosquitoes. In five experiments, it varied from 2.7×10³ to 9.0×10³. The sterile sporozoites, harvested on day 19 or 20 after the infective blood meal, were as infective for rodents as nonsterile ones.Supported in part by Public Health Service research grant AI 18345 from the National Institute of Allergy and Infectious Diseases, by a grant from the Agency of International Development DSPE-5542-G-SS-3042-00, and by a Charles and Johanna Busch award.     

Development of malaria parasites in mosquitoes fed with ookinetes suspended in defined media

Information concerning the specific nutritional requirements of malarial parasites developing in the mosquito host has been difficult to obtain, owing primarily to the complex nature of the blood meal that accompanies the parasites and the lack of success in culturing the complete invertebrate cycle of Plasmodium in vitro. The present report describes a blood-free system for infecting mosquitoes with ookinetes of Plasmodium berghei and for allowing the latter to develop into infective sporozoites. Ookinetes cultured in vitro were separated from blood proteins, suspended in defined medium, and fed to Anopheles stephensi mosquitoes through a membrane. The mosquitoes were then maintained on the same defined medium plus 5% sucrose. Infectivity of the parasites was demonstrated 17-19 days later by intracardial inoculation of the macerated mosquitoes into hamsters. This system makes it possible to evaluate nutritional factors that affect parasite development in the mosquito host under controlled conditions.