PREreview of Mitochondrial ATP synthesis is essential for efficient gametogenesis inPlasmodium falciparum

In the manuscript by Sparkes et al they investigate the role of mitochondrial ATP synthesis for gametogenesis in Plasmodium falciparum parasite. Due to biological differences between asexual stage and gametocyte stage parasites, mainly antimalarials are ineffective against gametocyte parasites leaving them free to continue their life-cycle within the mosquito.  Thus, there is a pressing need to further examine this critical life-stage for development of novel transmission blocking tools. Here, the authors first identify by transcriptional analysis a male-specific stage marker LDH2 for late stage IV gametocytes and subsequently validate the marker through immunofluorescence assays. They next contrasted the differences between nuclear and mitochondrial organization between male and female gametocytes, finding that male gametocytes have larger nuclear morphology while females have higher mitochondrial activity. They then used different mitochondrial inhibitors to determine the impact on the male/females during gametogenesis. As males show large inhibition upon compound treatment, they investigated the necessity of active mitochondrial ATP synthesis during male gametogenesis for exflagellation using FRET-based assay. Overall, the manuscript is well written. The introduction section contains enough background information for a new reader to understand P. falciparum gametocyte biology and states the gap in the field that is to be addressed. The methods (use of a transgenic parasite lines for the promoter experiments, Mitotracker for the impact on mitochondrial activity, and Dual Gamete Formation Assay for male and female gametogenesis) are correctly used in the study. The results section follows a logical progression into each experiment, using RNA-seq data to identify male specific promoters, using that information to test different mitochondrial inhibitors against male and females gametocytes, and then finally the impact of drug treatment under different media conditions (with glucose and glucose repleted media). Collectively, the identification of LDH2 as a molecular marker for male gametocytes and the defined role of ATP-synthesis required for male gametogenesis is a significant finding and supports the authors major conclusions in this study. The discussion section starts by summarizing the manuscript's main findings and then leads into a discussion on the different potential possibilities to address the complex question on the necessity of ATP generation for gametogenesis despite the absence of a mitochondrion in male gametes. The final section than details a path forward with future project goals in male/female gametocyte biology.

Major Points:

The results for Figure 5 examining the optimal media conditions going forward for the contribution of ATP from glycolysis or oxidative phosphorylation are interesting. Could this question not also be addressed by using chemical inhibitors of glycolysis? It would be insightful (and a much simpler way to test their hypothesis) It would be beneficial to have an additional compound (atovaquone or another ATP-synthase inhibitor) used in the male gametogenesis in Figure 6 to further support their findings. Although oligomycin A is an inhibitor of the ATP-synthase, which is what the authors would like to test, the Figure 4 results for the mitotracker and EC50 curves for the female gametocytes may indicate additional

Minor Points:

A few sentences are required to explain the methodology behind the readout for the Dual Gamete Formation Assay (i.e. what is Pfs25) for readers who are not familiar with the previous publication. Similarly, a schematic model of the design of the FRET system would be a great addition to Figure 7. The model shown in Figure 8 is helpful for summarizing the main findings of the manuscript for the reader, but it is confusing the three small circles representing the arrested male gametocytes all look the same. I believe the authors are trying to state the different nutrient conditions cause the arrest to occur at different points in development (the male gametocytes in the glucose-repleted sample with the mitochondrial ATP synthesis inhibitor seems to still develop 1-2 flagellum, while the other conditions in no glucose arrest much earlier); but this is lost when the representations all look similar in the figure. 

Competing interests

The author declares that they have no competing interests.