Gametocytes are essential for human-to-mosquito transmission of the malaria parasite Plasmodium falciparum, but despite their importance, many aspects of their cell biology remain poorly understood, including energy metabolism. It is known that gametocytes upregulate mitochondrial energy metabolism, but not how this is regulated. In addition, it has not previously been investigated whether energy metabolism differs between male and female gametocytes. Quantitative image analysis of functional mitochondrial labelling demonstrated that male gametocyte mitochondria are less active than those of females. Susceptibility of male and female gametocytes to mitochondrial electron transport chain (mETC) inhibitors was compared by two readouts: mitochondrial activity (MitoTracker labelling) and gamete formation (dual-gamete formation assay). Changes in mitochondrial activity were found to result in arrest of gametogenesis. Addition of oligomycin A with no pre-incubation also markedly reduced gametogenesis, suggesting that mitochondrial respiration is essential for optimal gametogenesis. Parasites expressing ATP nanosensors revealed that in excess glucose, intracellular ATP concentrations in gametocytes are insensitive to oligomycin A, demonstrating that glycolysis is sufficient to meet the energy needs of mature gametocytes. This supports the notion that upregulation of mitochondrial energy metabolism is to meet the energy demands of gametogenesis in males and onward mosquito stage development in females. Data mining of published proteomic data and bioinformatics approaches identified six proteins of interest, two of which were taken forward for characterisation. One of these proteins, a putative ADP-forming acetyl-CoA synthetase (ACS-ADP), was found to be mitochondrially localised and expressed throughout the life cycle. A second protein, phosphofructokinase 11 (PFK11), was successfully disrupted using CRISPR gene editing, resulting in a marked reduction in oocyst production, suggesting it is critical for ookinete development into oocysts. While further experiments are required to determine their exact function, this work has identified several potential regulators of gametocyte energy metabolism.
