Mitomycin-C is an unreliable inhibitor for study of DNA synthesis in Plasmodium.

Cytophotometric studies on DNA synthesis during asexual and sexual development of Plasmodium berghei contradicted earlier conclusions on DNA synthesis in Plasmodium which were largely based on experiments in which mitomycin-C had been used as a DNA replication inhibitor. Therefore, the effect of mitomycin on intra erythrocytic asexual development and on microgametogenesis, fertilization and zygote/ookinete development of P. berghei was studied in vitro. All DNA-synthesizing stages (schizonts, exflagellating microgametocytes and zygotes) and also DNA synthesis itself in all such stages, are totally unaffected by mitomycin concentrations 10 times higher than that which inhibits normal development of the non-DNA-synthesizing rings and trophozoites. The results are explained by the mode of action of mitomycin.

DNA synthesis in Plasmodium berghei during asexual and sexual development.

DNA contents of individual stages of Plasmodium berghei were measured by direct microfluorometry after Feulgen-pararosaniline (SO2) staining. Sporozoites, intra-erythrocytic ringforms and trophozoites (until at least 15 h after invasion) are haploid and non-synthesizing DNA. DNA is synthesized just before and during schizogony, which takes 4-6 h. Genome duplication and segregation are alternating events throughout this process. Mature micro- and macrogametocytes have DNA contents between the haploid and diploid value; most, if not all of the DNA in excess of the haploid value is synthesized during the last 5-10 h of maturation. During gametogenesis microgametocytes within 8-10 min synthesize DNA steadily and at a very high rate to more than the octoploid value while the DNA content of macrogametocytes remains constant. Fertilization in vitro takes place within 1 h after gamete formation. Within 2 h and coinciding with the onset of meiosis the zygote then synthesizes DNA up to almost the tetraploid value, after which synthesis stops during ookinete development. All the above mentioned processes of DNA synthesis are reversibly inhibited by aphidicolin (C50 from 3-13 microM). From the rate of DNA synthesis during microgametogenesis we calculated a minimum of 1300 origins of replication in the haploid genome of P. berghei.

Development of Plasmodium berghei ookinetes in the midgut of Anopheles atroparvus mosquitoes and in vitro.

Plasmodium berghei ookinete formation in vitro and within the midgut of susceptible Anopheles atroparvus were compared. No significant morphological differences were seen, except that in vitro development was more synchronized and less degenerating forms occurred. In vitro ookinete yields were 4-31 times higher and less variable than those in vivo. Mosquitoes of a susceptible and of a refractory line of A. atroparvus were simultaneously fed on the same host or via a membrane with the same suspension of in vitro-formed mature ookinetes. Up to 100% of mosquitoes of the susceptible line produced oocysts, mostly in high numbers, whereas infection rates and numbers of oocysts produced in mosquitoes of the refractory line were lower and much more so after host feeding than after membrane feeding of mature ookinetes, indicating that refractoriness does not depend on a single process of inhibition.

In vitro formation of ookinetes and functional maturity of Plasmodium berghei gametocytes.

In vitro formation of Plasmodium berghei ookinetes was studied. Gametocytes produced in vitro were obtained from heart and tail blood of Swiss mice and from blood removed from mosquitoes directly after feeding on these mice. In vitro produced gametocytes were obtained from short-term cultures of the erythrocytic stages of P. berghei. Reproducible ookinete production was obtained in medium RPMI 1640, pH 7.8-8.0, using in vivo and in vitro produced gametocytes. The morphology of developmental stages of ookinetes and degenerate forms at the light microscope level is described. More ookinetes were produced in medium RPMI 1640 compared to MEM and ookinete yield--defined as the ratio between the number of in vitro produced ookinetes/10(5) erythrocytes and the number of exflagellations/10(5) erythrocytes in the infected blood--increased with lower erythrocyte densities in the cultures within the range of dilutions tested. A linear relationship existed between gametocytaemia and the number of ookinetes produced. The methods for in vitro ookinete formation and for estimating ookinete yields enabled us to study aspects of functional maturity of gametocytes independent of mosquitoes. The numbers of exflagellating gametocytes and in vitro ookinete yields in tail blood corresponded with those in heart blood and blood ingested by mosquitoes, suggesting a random distribution of functionally mature gametocytes within the vertebrate host.