This is a proposal to study the developmental biology of the mosquito stages of the malaria parasite in vitro using invertebrate cell cultures as substrates. A variety of invertebrate cell lines and types will be evaluated for their ability to support sporogony but mosquito cell lines will be emphasized.
Our aim i s to study the transformation of ookinetes to oocysts and the differentiation of the sporozoite. Plasmodium berghei maintained in hamsters and Anopheles stephensi will be used. Axenic mosquitoes will be reared using mosquito cell cultures as larval food. Parasites will be harvested and concentrated from the infected mosquitoes and inoculated into cell cultures. Some studies will use ookinetes from cultures of infected hamster erythrocytes. We will develop the preparative procedures to obtain parasites in the numbers and purity needed for seeding replicate cultures. The morphogenesis, nutritional requirements and metabolism of the sporogonic stages will be studied using autoradiography, microscopical and biochemical techniques. Viability of the cultured parasites will be assessed using the criteria of motility, ability to incorporate radio labelled precursors of proteins and nucleic acids, and ability to continue development when placed back into the mosquito. The utilization of amino acids and carbohydrates in cultures infected with the sporogonic stages will be studied. The time sequence in the differentiation of sporozoite antigens and the acquisition of infectivity will be examined using monoclonal antibodies that recognize sporozoite antigens. The ultimate goal of the project is to develop culture systems that permit the continuing development of the sporogonic stages and the production of sporozoites from cyclically growing merozoite cultures. The information gained will be of value to immunologists for the development of a sporozoite vaccine against malaria.
