Gametocyte Sex Ratio: The Key to Understanding Plasmodium falciparum Transmission?

Recent RNA sequencing studies have uncovered a number of P. falciparum gametocyte sex-specific targets and provided new insights in gametocyte biology. After decades when gametocyte sex-ratio research was restricted to nonhuman malarias or in vitro experiments, molecular tools for assessing gametocyte sex ratio are now increasingly available for use in natural P. falciparum infections. Evidence that gametocyte sex ratio is influenced by total gametocyte density and antimalarial treatment, and improves predictions of transmission potential, highlight the relevance of understanding the gametocyte sex ratio during natural infections. The finding that the most widely used P. falciparum gametocyte marker Pfs25 is expressed predominantly by female gametocytes and has non-negligible levels of background expression in asexual parasites necessitates a re-evaluation of existing gametocyte data. A mosquito needs to ingest at least one male and one female gametocyte to become infected with malaria. The sex of Plasmodium falciparum gametocytes can be determined microscopically but recent transcriptomics studies paved the way for the development of molecular methods that allow sex-ratio assessments at much lower gametocyte densities. These sex-specific gametocyte diagnostics were recently used to examine gametocyte dynamics in controlled and natural infections as well as the impact of different antimalarial drugs. It is currently unclear to what extent sex-specific gametocyte diagnostics obviate the need for mosquito feeding assays to formally assess transmission potential. Here, we review recent and historic assessments of gametocyte sex ratio in relation to host and parasite characteristics, treatment, and transmission potential. A mosquito needs to ingest at least one male and one female gametocyte to become infected with malaria. The sex of Plasmodium falciparum gametocytes can be determined microscopically but recent transcriptomics studies paved the way for the development of molecular methods that allow sex-ratio assessments at much lower gametocyte densities. These sex-specific gametocyte diagnostics were recently used to examine gametocyte dynamics in controlled and natural infections as well as the impact of different antimalarial drugs. It is currently unclear to what extent sex-specific gametocyte diagnostics obviate the need for mosquito feeding assays to formally assess transmission potential. Here, we review recent and historic assessments of gametocyte sex ratio in relation to host and parasite characteristics, treatment, and transmission potential. The transmission of malaria from human to mosquito depends on the presence of gametocytes (see Glossary), sexual-stage Plasmodium parasites, in the peripheral blood. The development of gametocytes of P. falciparum takes 8–12 days and it involves transitions from committed rings, via early and intermediate stage gametocytes that are sequestered in the bone marrow and spleen (stages I–IV) [1Farfour E. et al.The extravascular compartment of the bone marrow: a niche for Plasmodium falciparum gametocyte maturation?.Malar. J. 2012; 11: 285Crossref PubMed Scopus (0) Google Scholar, 2Aguilar R. et al.Severity of anaemia is associated with bone marrow haemozoin in children exposed to Plasmodium falciparum.Br. J. Haematol. 2014; 164: 877-887Crossref PubMed Scopus (15) Google Scholar, 3Joice R. et al.Plasmodium falciparum transmission stages accumulate in the human bone marrow.Sci. Transl. Med. 2014; 6 (244re5)Crossref PubMed Scopus (81) Google Scholar], to mature male and female stage V gametocytes [4Sinden R. Malaria, sexual development and transmission: retrospect and prospect.Parasitology. 2009; 136: 1427-1434Crossref PubMed Scopus (31) Google Scholar]. In other Plasmodium species, such as P. vivax, P. chabaudi, P. vinckei, and P. gallinaceum, gametocytes develop much more rapidly, typically within 3 days [5Meibalan E. Marti M. Biology of malaria transmission.Cold Spring Harb. Perspect. Med. 2017; 7 (a025452)Crossref PubMed Scopus (10) Google Scholar]. In controlled human malaria infections (CHMIs) less than 10% of all asexual P. falciparum parasites commit to form gametocytes [6Collins K.A. et al.A controlled human malaria infection model enabling evaluation of transmission-blocking interventions.J. Clin. Invest. 2018; 128: 1551-1562Crossref PubMed Scopus (2) Google Scholar, 7Reuling I.J. et al.A randomized feasibility trial comparing four antimalarial drug regimens to induce Plasmodium falciparum gametocytemia in the controlled human malaria infection model.eLife. 2018; 7e31549Crossref PubMed Scopus (1) Google Scholar], and mature gametocytes typically comprise less than 5% of the circulating parasite biomass in natural infections [8Taylor L. Read A. Why so few transmission stages? Reproductive restraint by malaria parasites.Parasitol. Today. 1997; 13: 135-140Abstract Full Text PDF PubMed Scopus (123) Google Scholar]. Sexual commitment happens before the stage of schizogony [9Eichner M. et al.Genesis, sequestration and survival of Plasmodium falciparum gametocytes: parameter estimates from fitting a model to malaria therapy data.Trans. R. Soc. Trop. Med. Hyg. 2001; 95: 497-501Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 10Sinden R. Sexual development of malarial parasites.Adv. Parasitol. 1983; 22: 153-216Crossref PubMed Google Scholar, 11Poran A. et al.Single-cell RNA sequencing reveals a signature of sexual commitment in malaria parasites.Nature. 2017; 551: 95-99Crossref PubMed Scopus (0) Google Scholar, 12Bruce M.C. et al.Commitment of the malaria parasite Plasmodium falciparum to sexual and asexual development.Parasitology. 1990; 100: 191-200Crossref PubMed Google Scholar], and all merozoites derived from one schizont develop into either microgametocytes (males) or macrogametocytes (females) [13Silvestrini F. et al.Commitment to the production of male and female gametocytes in the human malaria parasite Plasmodium falciparum.Parasitology. 2000; 121: 465-471Crossref PubMed Scopus (66) Google Scholar, 14Smith T.G. et al.Commitment to sexual differentiation in the human malaria parasite, Plasmodium falciparum.Parasitology. 2000; 121: 127-133Crossref PubMed Scopus (73) Google Scholar]. The lack of sex chromosomes in haploid Plasmodium hampers our understanding of the commitment to sexual differentiation and the timing of sex determination [15Smith T.G. et al.Sexual differentiation and sex determination in the Apicomplexa.Trends Parasitol. 2002; 18: 315-323Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Sex determination could occur at the same moment when commitment to sexual differentiation is determined [13Silvestrini F. et al.Commitment to the production of male and female gametocytes in the human malaria parasite Plasmodium falciparum.Parasitology. 2000; 121: 465-471Crossref PubMed Scopus (66) Google Scholar], or alternatively via a two-stage process in which sex determination happens after the decision on commitment to sexual differentiation. At the molecular level, the nuclear protein P. falciparum gametocyte development 1 (GDV1) triggers the first known part of the molecular cascade of commitment and acts as the upstream regulator of the DNA-binding protein AP2-G (PF3D7_1222600), by antagonizing heterochromatin protein 1 repression of AP2-G transcription [16Filarsky M. et al.GDV1 induces sexual commitment of malaria parasites by antagonizing HP1-dependent gene silencing.Science. 2018; 359: 1259-1263Crossref PubMed Scopus (9) Google Scholar]. Sufficient activation of AP2-G, the master transcriptional regulator of gametocytogenesis, represents the 'point of no return' in commitment to both male and female gametocytes [11Poran A. et al.Single-cell RNA sequencing reveals a signature of sexual commitment in malaria parasites.Nature. 2017; 551: 95-99Crossref PubMed Scopus (0) Google Scholar, 17Kafsack B.F. et al.A transcriptional switch underlies commitment to sexual development in malaria parasites.Nature. 2014; 507: 248-252Crossref PubMed Scopus (139) Google Scholar, 18Sinha A. et al.A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium.Nature. 2014; 507: 253-257Crossref PubMed Scopus (138) Google Scholar]. The molecular basis of sex determination, and its timing during development, is currently unknown. Here, we summarize historic and recent estimates of gametocyte sex ratio in natural infections and the limitations of older estimates that relied on microscopy. We present the merits and limitations of molecular tools for quantifying gametocyte sex ratio and review evidence for a sex-specific effect of antimalarial drugs on circulating gametocytes and the implications for malaria transmission potential. In natural infections of P. falciparum, the sex ratio of circulating gametocytes is mostly female biased [19Bousema T. Drakeley C. Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination.Clin. Microbiol. Rev. 2011; 24: 377-410Crossref PubMed Scopus (343) Google Scholar]. Only a few epidemiological studies have quantified female and male gametocytes separately with sex ratios ranging from a mean of ∼three to five females to one male (Table 1) [20Delves M.J. et al.Male and female Plasmodium falciparum mature gametocytes show different responses to antimalarial drugs.Antimicrob. 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Evidence for environmental modulation of gametocytogenesis in Plasmodium falciparum in continuous culture.Bull. World Health Organ. 1979; 57: 37-52PubMed Google Scholar] (Figure 1). Gametocyte sex determination by microscopy is challenging due to the typically low gametocyte densities and the difficulty of allocating a sex to the sparsely observed gametocytes [22Robert V. et al.Effect of gametocyte sex ratio on infectivity of Plasmodium falciparum to Anopheles gambiae.Trans. R. Soc. Trop. Med. Hyg. 1996; 90: 621-624Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 25Schneider P. et al.Submicroscopic Plasmodium falciparum gametocyte densities frequently result in mosquito infection.Am. J. Trop. Med. Hyg. 2007; 76: 470-474Crossref PubMed Google Scholar, 26Coleman R.E. et al.Infectivity of asymptomatic Plasmodium-infected human populations to Anopheles dirus mosquitoes in western Thailand.J. Med. 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As an illustration of their sparseness, in a recent detailed study on gametocyte carriage in Burkina Faso, only 20% (95% confidence interval [CI] 14–27%) of patent asexual parasite carriers had microscopically detected gametocytes, and only 7% (95% CI 4–12%) had more than two gametocytes observed whilst enumerating against a conventional number of 500 white blood cells (∼0.06 μl of blood microscopically screened) [29Goncalves B.P. et al.Examining the human infectious reservoir for Plasmodium falciparum malaria in areas of differing transmission intensity.Nat. Commun. 2017; 8: 1133Crossref PubMed Scopus (25) Google Scholar]. The implications of low observed gametocyte counts for sex ratio precision are presented in Figure 1, indicating that observations based on 500 white blood cells can rarely lead to accurate gametocyte sex ratio estimates. Only by observing 50–100 gametocytes can a reproducible sex ratio be calculated (Figure 1D) [8Taylor L. Read A. Why so few transmission stages? Reproductive restraint by malaria parasites.Parasitol. Today. 1997; 13: 135-140Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 19Bousema T. Drakeley C. Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination.Clin. Microbiol. Rev. 2011; 24: 377-410Crossref PubMed Scopus (343) Google Scholar], and this is hardly ever attainable by routine microscopy. Microscopic investigation of gametocyte sex can be improved by concentrating gametocytes with magnetic enrichment [30Karl S. et al.Enhanced detection of gametocytes by magnetic deposition microscopy predicts higher potential for Plasmodium falciparum transmission.Malar. J. 2008; 7: 66Crossref PubMed Scopus (35) Google Scholar], assuming it enriches male and female gametocytes equally, allowing more gametocytes to be examined. The accuracy of differentiating male and female gametocytes can be further improved by targeting proteins or transcripts that are preferentially expressed in a specific sex using immunofluorescence or probe-based hybridization assays, respectively. Assays that target stage-specific expression of transcripts, such as Pf77 (PF3D7_0621400) and Pfg377 (PF3D7_1250100), both enriched in female gametocytes [31Baker D.A. et al.Sexual-stage-specific RNA expression of a new Plasmodium falciparum gene detected by in situ hybridisation.Mol. Biochem. Parasitol. 1995; 72: 193-201Crossref PubMed Google Scholar, 32Alano P. et al.COS cell expression cloning of Pfg377, a Plasmodium falciparum gametocyte antigen associated with osmiophilic bodies.Mol. Biochem. Parasitol. 1995; 74: 143-156Crossref PubMed Scopus (64) Google Scholar] and Pfg27 (PF3D7_1302100, detecting both sexes) [33Olivieri A. et al.The Plasmodium falciparum protein Pfg27 is dispensable for gametocyte and gamete production, but contributes to cell integrity during gametocytogenesis.Mol. Microbiol. 2009; 73: 180-193Crossref PubMed Scopus (21) Google Scholar, 34Stone W. et al.A molecular assay to quantify male and female Plasmodium falciparum gametocytes: results from 2 randomized controlled trials using primaquine for gametocyte clearance.J. Infect. Dis. 2017; 216: 457-467Crossref PubMed Scopus (17) Google Scholar] have been reported previously. Immunofluorescence assays based on antibodies that bind proteins specific for early gametocytes such as Pfs16 (PF3D7_0406200) [14Smith T.G. et al.Commitment to sexual differentiation in the human malaria parasite, Plasmodium falciparum.Parasitology. 2000; 121: 127-133Crossref PubMed Scopus (73) Google Scholar, 35Bruce M.C. et al.Cellular location and temporal expression of the Plasmodium falciparum sexual stage antigen Pfs16.Mol. Biochem. Parasitol. 1994; 65: 11-22Crossref PubMed Scopus (73) Google Scholar], male gametocytes (α-tubulin II, PF3D7_0422300) [13Silvestrini F. et al.Commitment to the production of male and female gametocytes in the human malaria parasite Plasmodium falciparum.Parasitology. 2000; 121: 465-471Crossref PubMed Scopus (66) Google Scholar, 14Smith T.G. et al.Commitment to sexual differentiation in the human malaria parasite, Plasmodium falciparum.Parasitology. 2000; 121: 127-133Crossref PubMed Scopus (73) Google Scholar, 36Rawlings D.J. et al.α-Tubulin II is a male-specific protein in Plasmodium falciparum.Mol. Biochem. Parasitol. 1992; 56: 239-250Crossref PubMed Scopus (0) Google Scholar], or female gametocytes (Pfg377, PF3D7_1250100) [13Silvestrini F. et al.Commitment to the production of male and female gametocytes in the human malaria parasite Plasmodium falciparum.Parasitology. 2000; 121: 465-471Crossref PubMed Scopus (66) Google Scholar] have also been used. Insights into sex differences in the transcriptional and proteomic makeup are summarized in Box 1. These immunofluorescent assays alleviate common problems that complicate microscopic examination but their reliance on fluorescence microscopy greatly affects their deployment in the field. The recent development of molecular assays to quantify male and female gametocytes may allow more robust sex ratio determination at low gametocyte densities and easier application in field settings.Table 1Summary of Studies That Evaluated Sex Ratio in Natural InfectionsSettingPopulationProportion maleaProportion male is defined as the proportion of all gametocytes that is male, [male gametocytes/(male+female gametocytes)].ToolRefsMali, Burkina Faso, CameroonAsymptomatic gametocyte carriers0.14–0.51qRT-PCR23Bradley J. et al.Predicting the likelihood and intensity of mosquito infection from sex specific Plasmodium falciparum gametocyte density.eLife. 2018; 7e34463Crossref PubMed Scopus (4) Google ScholarThe NetherlandsControlled human malaria infection volunteers0.29qRT-PCR7Reuling I.J. et al.A randomized feasibility trial comparing four antimalarial drug regimens to induce Plasmodium falciparum gametocytemia in the controlled human malaria infection model.eLife. 2018; 7e31549Crossref PubMed Scopus (1) Google ScholarMaliAsymptomatic gametocyte carriers0.30qRT-PCR62Dicko A. et al.Efficacy and safety of primaquine and methylene blue for prevention of Plasmodium falciparum transmission in Mali: a phase 2, single-blind, randomised controlled trial.Lancet Infect. Dis. 2018; 18: 627-639Abstract Full Text Full Text PDF PubMed Scopus (13) Google ScholarAustraliaControlled human malaria infection volunteers0.20qRT-PCR6Collins K.A. et al.A controlled human malaria infection model enabling evaluation of transmission-blocking interventions.J. Clin. Invest. 2018; 128: 1551-1562Crossref PubMed Scopus (2) Google ScholarKenya, MaliAsymptomatic gametocyte carriers0.36qRT-PCR34Stone W. et al.A molecular assay to quantify male and female Plasmodium falciparum gametocytes: results from 2 randomized controlled trials using primaquine for gametocyte clearance.J. Infect. Dis. 2017; 216: 457-467Crossref PubMed Scopus (17) Google ScholarNigeriaSymptomatic malaria patients0.21Microscopy75Gbotosho G.O. et al.Plasmodium falciparum gametocyte carriage, sex ratios and asexual parasite rates in Nigerian children before and after a treatment protocol policy change instituting the use of artemisinin-based combination therapies.Mem. Inst. Oswaldo Cruz. 2011; 106: 685-690Crossref PubMed Scopus (0) Google ScholarNigeriaSymptomatic children0.34Microscopy87Gbotosho G.O. et al.Plasmodium falciparum gametocyte carriage, emergence, clearance and population sex ratios in anaemic and non-anaemic malarious children.Mem. Inst. Oswaldo Cruz. 2011; 106: 562-569Crossref PubMed Google ScholarNigeriaSymptomatic malaria patients0.22Microscopy88Sowunmi A. et al.Population structure of Plasmodium falciparum gametocyte sex ratios in malarious children in an endemic area.Parasitol. Int. 2009; 58: 438-443Crossref PubMed Scopus (11) Google ScholarNigeriaSymptomatic malaria patients0.75Microscopy76Sowunmi A. et al.Effects of mefloquine and artesunate mefloquine on the emergence, clearance and sex ratio of Plasmodium falciparum gametocytes in malarious children.Malar. J. 2009; 8: 297Crossref PubMed Scopus (10) Google ScholarIndiaSymptomatic malaria patients0.31Microscopy89Kar P. et al.Plasmodium falciparum gametocytaemia with chloroquine chemotherapy in persistent malaria in an endemic area of India.Indian J. Med. Res. 2009; 129: 299-305PubMed Google ScholarNigeriaSymptomatic malaria patients0.20Microscopy78Sowunmi A. et al.Plasmodium falciparum gametocyte sex ratios in children with acute, symptomatic, uncomplicated infections treated with amodiaquine.Malar. J. 2008; 7: 169Crossref PubMed Scopus (0) Google ScholarNigeriaSymptomatic malaria patients0.05Microscopy90Sowunmi A. et al.Activities of artemether-lumefantrine and amodiaquine-sulfalene-pyrimethamine against sexual-stage parasites in falciparum malaria in children.Chemotherapy. 2008; 54: 201-208Crossref PubMed Scopus (13) Google ScholarNigeriaSymptomatic malaria patients0.14Microscopy91Sowunmi A. et al.Activities of amodiaquine, artesunate, and artesunate-amodiaquine against asexual- and sexual-stage parasites in falciparum malaria in children.Antimicrob. Agents Chemother. 2007; 51: 1694-1699Crossref PubMed Scopus (0) Google ScholarSenegalSymptomatic children0.15Microscopy92Talman A.M. et al.Influence of chemotherapy on the Plasmodium gametocyte sex ratio of mice and humans.Am. J. Trop. Med. Hyg. 2004; 71: 739-744Crossref PubMed Google ScholarSenegalTotal population0.35Microscopy93Robert V. et al.Sex ratio of Plasmodium falciparum gametocytes in inhabitants of Dielmo, Senegal.Parasitology. 2003; 127: 1-8Crossref PubMed Scopus (0) Google ScholarNigeriaAsymptomatic children0.42Microscopy77Sowunmi A. Fateye B.A. Gametocyte sex ratios in children with asymptomatic, recrudescent, pyrimethamine-sulfadoxine-resistant, Plasmodium falciparum malaria.Ann. Trop. Med. Parasitol. 2003; 97: 671-682Crossref PubMed Scopus (15) Google ScholarTanzaniaNot applicablebInbreeding coefficients calculated directly from oocyst selfing rates from mosquitoes.0.34Indirect: calculated from inbreeding coefficient94Babiker H.A. et al.Genetic structure and dynamics of Plasmodium falciparum infections in the Kilombero region of Tanzania.Trans. R. Soc. Trop. Med. Hyg. 1999; 93: 11-14Abstract Full Text PDF PubMed Google ScholarThe GambiaNot applicablecInbreeding coefficients calculated from allele frequencies in blood-stage infections, but sex ratios not determined in these patients. Patients were symptomatic children.0.22Indirect: calculated from inbreeding coefficient94Babiker H.A. et al.Genetic structure and dynamics of Plasmodium falciparum infections in the Kilombero region of Tanzania.Trans. R. Soc. Trop. Med. Hyg. 1999; 93: 11-14Abstract Full Text PDF PubMed Google ScholarSudanNot applicabledInbreeding coefficients calculated from allele frequencies in blood-stage infections, but sex ratios not determined in these patients. Patients were symptomatic children and adults.0.07Indirect: calculated from inbreeding coefficient94Babiker H.A. et al.Genetic structure and dynamics of Plasmodium falciparum infections in the Kilombero region of Tanzania.Trans. R. Soc. Trop. Med. Hyg. 1999; 93: 11-14Abstract Full Text PDF PubMed Google ScholarCameroonSymptomatic malaria patients0.22Microscopy22Robert V. et al.Effect of gametocyte sex ratio on infectivity of Plasmodium falciparum to Anopheles gambiae.Trans. R. Soc. Trop. Med. Hyg. 1996; 90: 621-624Abstract Full Text PDF PubMed Scopus (87) Google ScholarPapua New GuineaNot applicablebInbreeding coefficients calculated directly from oocyst selfing rates from mosquitoes.0.04Indirect: calculated from inbreeding coefficient95Paul R.E. et al.Mating patterns in malaria parasite populations of Papua New Guinea.Science. 1995; 269: 1709-1711Crossref PubMed Google ScholarPapua New GuineaNot indicated0.18Microscopy96Read A.F. et al.Gametocyte sex ratios as indirect measures of outcrossing rates in malaria.Parasitology. 1992; 104: 387-395Crossref PubMed Google Scholara Proportion male is defined as the proportion of all gametocytes that is male, [male gametocytes/(male + female gametocytes)].b Inbreeding coefficients calculated directly from oocyst selfing rates from mosquitoes.c Inbreeding coefficients calculated from allele frequencies in blood-stage infections, but sex ratios not determined in these patients. Patients were symptomatic children.d Inbreeding coefficients calculated from allele frequencies in blood-stage infections, but sex ratios not determined in these patients. Patients were symptomatic children and adults. Open table in a new tab Box 1The Development of Male and Female GametocytesMolecular mechanisms underlying the differentiation switch towards becoming male or female gametocytes remain largely unknown. Candidate genes that may be associated with sex-specific differentiation include the AP2-G2 in P. berghei [18Sinha A. et al.A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium.Nature. 2014; 507: 253-257Crossref PubMed Scopus (138) Google Scholar], the Puf family of translational repressors in P. falciparum [97Fan Q. et al.Characterization of PfPuf2, member of the Puf family RNA-binding proteins from the malaria parasite Plasmodium falciparum.DNA Cell Biol. 2004; 23: 753-760Crossref PubMed Scopus (24) Google Scholar, 98Miao J. et al.The Puf-family RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum.J. Cell Sci. 2010; 123: 1039-1049Crossref PubMed Scopus (45) Google Scholar], and MAPK1 and MAPK2 as potential regulators of female and male gametocytogenesis in P. falciparum, respectively [63Walzer K.A. et al.Single-cell analysis reveals distinct gene expression and heterogeneity in male and female Plasmodium falciparum gametocytes.mSphere. 2018; 3 (e00130-18)Crossref PubMed Scopus (3) Google Scholar].Male gametocytes are terminally differentiated forms with one last task ahead; producing motile microgametes upon activation. Proteomic and transcriptomic data reflect this, showing upregulation of genes involved in nucleic acid metabolism, DNA replication, and axoneme formation. Female gametocytes, on the other hand, contain many proteins and transcripts intended for longer term sustainment of postfertilization stages, including genes involved in protein biosynthesis, degradation, transport, and metabolic activity. Another important female gametocyte-specific mechanism is translational repression in which stored transcripts support cellular processes immediately after fertilization, when transcription is absent for multiple hours [99Paton M.G. et al.Structure and expression of a post-transcriptionally regulated malaria gene encoding a surface protein from the sexual stages of Plasmodium berghei.Mol. Biochem. Parasitol. 1993; 59: 263-275Crossref PubMed Scopus (135) Google Scholar, 100Thompson J. Sinden R.E. In situ detection of Pbs21 mRNA during sexual development of Plasmodium berghei.Mol. Biochem. Parasitol. 1994; 68: 189-196Crossref PubMed Scopus (0) Google Scholar]. The deletion of genes involved in the maintenance of translationally repressed mRNA transcripts, such as DOZI [101Mair G.R. et al.Regulation of sexual development of Plasmodium by translational repression.Science. 2006; 313: 667-669Crossref PubMed Scopus (266) Google Scholar] and CITH [102Mair G.R. et al.Universal features of post-transcriptional gene regulation are critical for Plasmodium zygote development.PLoS Pathog. 2010; 6e1000767Crossref PubMed Scopus (121) Google Scholar] in P. berghei, results in full developmental arrest during ookinete formation, although fertilization is successful. The first indication that translational repression is also active in P. falciparum was obtained through deletion of Puf2, which resulted in altered expression of genes known to be translationally repressed, such as Pfs25 and Pfs28 [103Turque O. et al.Translational repression in malaria sporozoites.Microb. Cell. 2016; 3: 227-229Crossref PubMed Google Scholar, 104Muller K. et al.The Puf-family RNA-binding protein Puf2 controls sporozoite conversion to liver stages in the malaria parasite.PLoS One. 2011; 6e19860Crossref PubMed Scopus (0) Google Scholar]. More recent proteomics and transcriptomics data have uncovered a large-scale translational repression of hundreds of transcripts in P. falciparum female gametocytes [41Lasonder E. et al.Integrated transcriptomic and proteomic analyses of P. falciparum gametocytes: molecular insight into sex-specific processes and translational repression.Nucleic Acids Res. 2016; 44: 6087-6101Crossref PubMed Scopus (49) Google Scholar]. Molecular mechanisms underlying the differentiation switch towards becoming male or female gametocytes remain largely unknown. Candidate genes that may be associated with sex-specific differentiation include the AP2-G2 in P. berghei [18Sinha A. et al.A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium.Nature. 2014; 507: 253-257Crossref PubMed Scopus (138) Google Scholar], the Puf family of translational repressors in P. falciparum [97Fan Q. et al.Characterization of PfPuf2, member of the Puf family RNA-binding proteins from the malaria parasite Plasmodium falciparum.DNA Cell Biol. 2004; 23: 753-760Crossref PubMed Scopus (24) Google Scholar, 98Miao J. et al.The Puf-family RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum.J. Cell Sci. 2010; 123: 1039-1049Crossref PubMed Scopus (45) Google Scholar], and MAPK1 and MAPK2 as potential regulators of female and male gametocytogenesis in P. falciparum, respectively [63Walzer K.A. et al.Single-cell analysis reveals distinct gene expression and heterogeneity in male and female Plasmodium falciparum gametocytes.mSphere. 2018; 3 (e00130-18)Crossref PubMed Scopus (3) Google Scholar]. Male gametocytes are terminally differentiated forms with one last task ahead; producing motile microgametes upon activation. Proteomic and transcriptomic data reflect this, showing upregulation of genes involv