Cross-fertilisation in the malaria parasite Plasmodium falciparum

The objective of this work has been to investigate the frequency of cross-fertilisation between gametes of genetically distinct clones of the human malaria parasite Plasmodium falciparum. Previous genetic experiments involving both rodent malaria parasites in vivo and human malaria parasites in vitro have demonstrated higher than expected numbers of recombinants among the progeny of crosses. It has been suggested that this could be due to a favouring of cross-fertilisation over self-fertilisation in the mosquito phase of the life-cycle. The work has involved examining the genotypes of individual oocysts (derived from individual zygotes) resulting from mixed infection of two clones in mosquitoes. In preliminary work using the mouse malaria parasite P. yoelii nigeriensis attempts were made to examine the chromosomes of single oocysts using pulsed field gradient gel electrophoresis. However there was insufficient DNA in an oocyst to allow chromosomes to be visualised using this technique. The bulk of the work has been concerned with P. falciparum. In the first stage oligonucleotide primers suitable for use in the polymerase chain reaction (PCR) were designed to allow amplification of repetitive regions of two polymorphic antigen genes, denoted MSP1 and MSP2. The two clones of P. falciparum used in the crossing experiments possessed a different allele of each gene. These alleles were found to be recognisable as size differences of the PCR-amplified fragments on agarose gels. Gametocytes of the two clones were grown in vitro. Mixtures of gametocytes of each clone were made and fed to Anopheles stephensi or A. gambiae mosquitoes through membrane feeders. 9 to 10 days later the mosquitoes were dissected and their midguts were examined for the presence of oocysts. Individual oocysts were dissected from the midguts and the DNA extracted from them.