Highly purified mitochondrial fractions have been isolated from the intraerythrocytic stages of two mammalian Plasmodium spp., Plasmodium yoelii of rodents and Plasmodium falciparum of man. Mitochondria of the former parasite are cristate whereas those of the latter are essentially acristate. Isolated mitochondria from both parasite species were heterogeneous with respect to size, shape, density of matrix staining and extent of internal structure. Respiratory assay, by reduction of exogenous cytochrome c, showed NADH, α-glycerophosphate and succinate to be the substrates with the greatest potential for metabolism. Additionally, proline, dihydroorotate and glutamate (P. falciparum only) were oxidized at low rates. A number of NAD+-linked substrates were not utilized. The NADH-dependent reduction of cytochrome c was insensitive to rotenone and antimycin A. Fumarate inhibited the NADH-dependent reduction of cytochrome c and stimulated the oxidation of NADH, suggestive of an NADH–fumarate reductase pathway. Oxidation of either α-glycerophosphate or succinate was fully inhibited by standard mitochondrial electron transport inhibitors, including a number of Complex III inhibitors, although the concentrations required of such inhibitors (notably myxothiazol) were relatively high compared to mammalian mitochondria. Dithionite-reduced minus oxidized difference spectra indicated the presence of cytochromes aa3, b, c and c1 in mitochondria of both parasite species, but at a higher cytochrome to protein ratio in P. yoelii. Freshly isolated mitochondria from either species exhibited only low respiratory control ratios with α-glycerophosphate or succinate as substrates. The apparent absence of a respiratory chain ‘Site I’ in such mitochondria may mean that NADH–fumarate reductase serves to reoxidize mitochondrial NADH.

The transport processes of D-glucose in Plasmodium yoelii-infected mouse erythrocytes were investigated using 2-deoxy-D-glucose (2DOG), a non-metabolizable analogue of D-glucose. Infected cells showed an increase in the uptake of 2DOG compared to uninfected controls, and an effect which was more prominent in cells with mature-stage parasites. Kinetic studies measuring the initial rates of 2DOG uptake revealed two components in infected cells with late trophozoite and schizont-stage parasites: a simple diffusion system and a carrier (transporter)-mediated system. The transporter was common for D-glucose and 2DOG and had a kinetic constant indicating a high affinity for 2DOG (the Km = 0·18 mM and the Vmax = 0·61 mmol/1010 cells/min), as compared to the constant of the mouse erythrocyte carrier (the Km = 10 mM and the Vmax = 1·8 mmol/1010 cells/min). Determination of the distribution of [3H]2DOG in infected cells and experiments with metabolic inhibitors indicated that the simple diffusion system localizes in the membrane of host cells and the transporter in the parasite plasma membrane. The parasite glucose transporter was much less sensitive to cytochalasin B than that of the host cells and the uptake of 2DOG via the transporter was dependent on energy. Based on these findings, the following features emerge: D-glucose first gains access to the cytosol of infected erythrocytes via the simple diffusion system, which appears after infection by the parasite, and an active uptake against the concentration gradient takes place at the parasite plasma membrane via the parasite glucose transporter in an energy dependent manner. Finally, an energy transduction mechanism for the transport of glucose across the parasite plasma membrane is discussed.

Cloned lines of the four rodent Plasmodium species can be differentiated by the RFLP pattern generated following Southern blotting and probing with PCsv4.1, a probe derived from a P. chabaudi chabaudi genomic library. Groups of CBA/Ca mice were inoculated simultaneously with cloned lines from two parasite species or strains. Six mixed species and three mixed strain infections using rodent malaria lines were initiated. The composition of the parasite population in each group was determined qualitatively and semi-quantitatively by analysis of the DNA purified from daily blood samples, thereby providing a dynamic representation of each mixed infection. Effects on the course of parasitaemias are presented and discussed.

In order to study the effects of acclimatization of Plasmodium in β-thalassaemic mice, we used a mouse model of β-thalassaemia (DBA/2J/β-thal/β-thal), similar to that observed in humans. We acclimatized 3 rodent malarias (P. berghei, P. chabaudi and P. yoelii) in DBA/2J and DBA/2J/β-thal/β-thal mice lines, by 4 intraperitoneal serial transfers. All 3 rodent malarias developed in red blood cells of β-thalassaemic mice without losing their virulence. There was no delay in infection and peaks of parasitaemia were similar in β-thalassaemic and normal mice. The mortality occurred earlier in β-thalassaemic mice than in control mice for P. berghei and P. chabaudi. This difference was more pronounced for P. yoelii NS where normal mice did not die. These results could be explained by a failure of erythropoiesis in β-thalassaemic mice, which are unable to compensate for the destruction of red blood cells by the parasites, and the mice died of anaemia. Ultrastructural examination of the rodent malaria parasites in β-thalassaemic RBC showed a normal development even in the presence of Heinz bodies. In conclusion, no effective protection against malaria was provided by the β-thalassaemia in this mouse model.

Plasmodium spp. cause the worst parasitic diseases in humans and evade host immunity in complicated ways. Activated catabolism of tryptophan in dendritic cells is thought to suppress immunity, which is mediated by an inducible rate-limiting enzyme of tryptophan catabolism, indoleamine 2,3 dioxygenase (IDO), via both tryptophan depletion and production of toxic metabolites. In various infections, including malaria, IDO is known to be activated but its biological significance is unclear; therefore, we investigated whether malaria parasites induce IDO to suppress host immune responses. We found that enzymatic activity of IDO was elevated systematically in our mouse malaria model, and was abolished by in vivo IDO inhibition with 1-methyl tryptophan. Experimental infection with Plasmodium yoelii showed that IDO inhibition slightly suppressed parasite density in association with enhanced proliferation and IFN-γ production by CD4+ T cells in response to malaria parasites. Our observations suggest that induction of IDO is one of the immune mechanisms of malaria parasites.

The Py235 merozoite rhoptry protein of the rodent malaria agent Plasmodium (yoelii) yoeli is encoded by the Py235 multigene family whose members are transcribed during the parasite's asexual life-cycle in a fashion where single schizonts subsequently give rise to sets of merozoites containing distinct Py235 transcripts. Homologues of Py235 are found in other malaria species, and antibodies to both Py235 and P. falciparum homologues inhibit merozoite invasion, suggesting a unique survival strategy involving immune evasion and host adaptation. Using a mathematical approach to model this free-living stage of Plasmodium in interaction with specific antibodies and a heterogeneous red blood cell population, we investigate if, and under what conditions, this mechanism of clonal phenotypic variation can play a role in immune evasion and adaptation to a dynamic erythropoietic environment.

Changes in the pattern of haemolymph polypeptides were studied to map the innate immune response of the malaria vector Anopheles stephensi (Diptera: Culicidae) to infection by the parasite Plasmodium yoelii yoelii. In addition, changes in the ontogenetic pattern due to glucose feeding, age, sex and gonotrophic cycle-related proteins were analysed. Six polypeptides were induced and 22 polypeptides disappeared due to glucose feeding during various stages of the mosquito's adult life. Five polypeptide patterns (91, 100, 108, 133 & 145 KDa) were found exclusively in female haemolymph. The changes in these polypeptide patterns have been correlated with sexual dimorphism in their feeding behaviour.

In total, 18 polypeptides were induced in the haemolymph of parasitised mosquitoes during different stages of development. Most of the polypeptides were induced in the early stages of infection, i.e. immediately after a bloodmeal. One polypeptide (25 KDa) was induced in all the stages. The expression of two polypeptides (32.5 and 70 KDa) on day 9 and one polypeptide (42 KDa) on day 15 was also enhanced following parasitism. The different induced proteins may help mosquitoes of different ages cope with parasite infection. The parasite evidently elicits immune responses in multiple tissues of the mosquito, including two epithelia which the parasite must traverse to complete its development. The mechanism of these responses and their significance in malaria transmission are discussed.