Variation in Plasmodium falciparum sexual commitment rates and responses to environmental modification

Asexual blood stage malaria parasites must produce sexual progeny in order to infect mosquitoes. It is therefore important to understand the scope of variation in sexual commitment rates of the most important human parasite Plasmodium falciparum, and to assess responses to conditions that may affect this commitment. First, using a genetically modified line with inducible elevated sexual commitment we compared two different methods of measuring the rates, either staining the Pfs16 marker in very early gametocytes or counting gametocytes after further development. The methods showed correlated estimates in evaluation across multiple independent replicate assays, with higher sensitivity and precision being achieved by Pfs16 marker detection in early gametocytes, so this method was used to survey a wide range of P. falciparum lines. A panel of six recently culture-established clinical isolates from Ghana showed mean sexual commitment rates per cycle ranging from 3.3% to 12.2%, with some significant differences among isolates as well as variation among biological replicates of each. Comparing 13 long-term laboratory-adapted lines of diverse origins, eight had sexual commitment rates similar to those of the recent clinical isolates, with means across multiple biological replicates for each line ranging from 4.7% to 14.5%, while four lines had significantly lower rates with means ranging from 0.3 to 1.6%, and one line with a non-functional ap2-g gene always showed zero sexual commitment. Among a subset of parasite lines, adding choline to suppress commitment had quantitatively variable effects, although these were significant in most assays of lines that had relatively high rates. This study indicates the importance of multiple assay replicates and comparisons of diverse isolates to understand natural and culture-induced variation in this key reproductive trait, relevant to investigating potential effects of parasite adaptation on transmission. Author Summary Only sexual malaria parasites are transmitted from humans to mosquitoes, so it is vital to understand variation in sexual commitment rates of blood stage malaria parasites, and responses to conditions that affect this. We compared two different methods of measuring the rates in a Plasmodium falciparum line with engineered sexual commitment variation, demonstrating higher sensitivity and precision by detection of an early differentiation marker, and this method was then used to survey diverse P. falciparum lines in culture with extensive biological replicate measurements. Recent clinical isolates from Ghana showed mean sexual commitment rates per cycle ranging from 3% to 12%, with significant differences among isolates as well as variation among biological replicates. Long-term laboratory-adapted lines of diverse origins had a wide range, most having rates similar to those of the clinical isolates, while a minority consistently had much lower or zero rates. There was quantitative variation among lines in the effects of adding choline to suppress commitment, although most assays of lines that had relatively high rates showed significant effects. Performing multiple biological replicates and comparisons of recent parasite isolates is vital to understand intra-specific variation in this important reproductive trait, and move towards investigating direct effects on disease transmission.


Abstract
Asexual blood stage malaria parasites must produce sexual progeny in order to infect mosquitoes. It is therefore important to understand the scope of variation in sexual commitment rates of the most important human parasite Plasmodium falciparum, and to assess responses to conditions that may affect this commitment. First, using a genetically modified line with inducible elevated sexual commitment we compared two different methods of measuring the rates, either staining the Pfs16 marker in very early gametocytes or counting gametocytes after further development. The methods showed correlated estimates in evaluation across multiple independent replicate assays, with higher sensitivity and precision being achieved by Pfs16 marker detection in early gametocytes, so this method was used to survey a wide range of P. falciparum lines. A panel of six recently culture-established clinical isolates from Ghana showed mean sexual commitment rates per cycle ranging from 3.3% to 12.2%, with some significant differences among isolates as well as variation among biological replicates of each. Comparing 13 long-term laboratoryadapted lines of diverse origins, eight had sexual commitment rates similar to those of the recent clinical isolates, with means across multiple biological replicates for each line ranging from 4.7% to 14.5%, while four lines had significantly lower rates with means ranging from 0.3 to 1.6%, and one line with a non-functional ap2-g gene always showed zero sexual commitment. Among a subset of parasite lines, adding choline to suppress commitment had quantitatively variable effects, although these were significant in most assays of lines that had relatively high rates. This study indicates the importance of multiple assay replicates and comparisons of diverse isolates to understand natural and culture-induced variation in this key reproductive trait, relevant to investigating potential effects of parasite adaptation on transmission.

Author Summary
Only sexual malaria parasites are transmitted from humans to mosquitoes, so it is vital to understand variation in sexual commitment rates of blood stage malaria parasites, and responses to conditions that affect this. We compared two different methods of measuring the rates in a Plasmodium falciparum line with engineered sexual commitment variation, demonstrating higher sensitivity and precision by detection of an early differentiation marker, and this method was then used to survey diverse P. falciparum lines in culture with extensive biological replicate measurements. Recent clinical isolates from Ghana showed mean sexual commitment rates per cycle ranging from 3% to 12%, with significant differences among isolates as well as variation among biological replicates. Long-term laboratory-adapted lines of diverse origins had a wide range, most having rates similar to those of the clinical isolates, while a minority consistently had much lower or zero rates.
There was quantitative variation among lines in the effects of adding choline to suppress commitment, although most assays of lines that had relatively high rates showed significant effects. Performing multiple biological replicates and comparisons of recent parasite isolates is vital to understand intra-specific variation in this important reproductive trait, and move towards investigating direct effects on disease transmission.

Introduction
For malaria to be transmitted, asexual blood stage parasites must undergo differentiation which leads to formation of male and female gametocytes that are required to infect mosquitoes. This process involves an initial parasite commitment phase, when the switch to sexual development is induced, followed by a longer developmental phase to produce the mature gametocyte forms. From an evolutionary or ecological perspective, it is important to understand what determines the rate of commitment to sexual forms [1], and how this may vary within a species [2]. It is particularly vital for understanding P. falciparum transmission as more effective control is needed to reduce the global malaria burden [3], which is particularly challenging as chronic asymptomatic infections are often highly infective to mosquitoes [4].
Research on malaria parasite sexual conversion has been long established [5], and progress has been made in elucidating key aspects of the mechanism. An ApiAP2-family transcription factor AP2-G is essential for sexual conversion, controlling expression of genes critical to early gametocytogenesis [6][7][8][9]. The ap2-g locus is clonally variably expressed, with histone 2 lysine 9 trimethylation (H3K9me3) marks of heterochromatin silencing initially indicating that its expression could be epigenetically regulated [10], and conditional depletion of heterochromatin protein 1 (HP1) from the promoter was shown to increase ap2-g transcription and sexual conversion [11]. Furthermore, in P. falciparum a gametocyte development protein GDV1, that was previously linked with sexual commitment [12], is involved in removal of HP1 from the ap2-g locus so that over-expression of gdv1 causes elevated sexual commitment rates [13].
The classic model is that sexual commitment occurs in the intra-erythrocytic cycle prior to the cycle in which parasite sexual differentiation occurs, based on a finding that in static culture individual schizonts of the 3D7 parasite clone yield spatially-clustered intraerythrocytic progeny that tend to differentiate similarly, either sexually or asexually [14].
However, there is recent evidence that some sexual commitment may also occur in the same cycle as sexual differentiation, if ap2-g expression is induced to start very early after erythrocyte invasion during the ring stage [15], although it is not known how commonly this occurs in parasites under normal conditions of infection or laboratory culture. It is difficult to study P. falciparum gametocyte conversion rates precisely in natural infections, as most stages of developing gametocytes do not occur in the peripheral circulation and are therefore not detected in blood samples, although circulating ring stage parasites that are sexually committed may become detectable by developing assays for early markers such as AP2-G or GEXP02 [16,17]. Correlations between P. falciparum gametocyte numbers and other measured variables in human hosts, such as age or hemoglobin levels, may not be causally related to varying gametocyte conversion rates and are not well replicated across different studies [18]. Experiments with other malaria parasite species in laboratory mice indicate sexual commitment to be a plastic phenotype that can respond to the in vivo environment [19]. There are few data relating to P. falciparum gametocyte conversion in experimentally induced human infections, but modelling of results from human experimental infections with the parasite clone 3D7 has indicated that the conversion rate per cycle is usually less than 1% [20], and older data on induced malaria infections with strains that no longer exist suggest broadly similar average rates [21].
Early studies of P. falciparum in culture indicated that gametocyte production varied among parasite clones [22,23], and also varied with culture conditions, although precise estimates of conversion rates per cycle were not established. Subsequent studies estimated gametocyte conversion rates of several laboratory-adapted P. falciparum clones to be usually above 1% per cycle when grown at low parasitaemia [6,24], with higher rates if parasites were stressed at high parasitaemia or by antimalarial drug treatment [14,24].
Parasites appear to respond to metabolic challenges during growth, although most cellular mechanisms remain to be identified [25]. An increase in gametocytogenesis may be triggered by cellular stress involving redox perturbation [26,27], by adding low doses of the antimalarial artemisinin at the early trophozoite growth stage [28], or by adding parasiteconditioned culture medium [29] or extracellular vesicles from such medium [30]. Notably, gametocyte conversion rates in cultured lines are suppressed by lysophosphatidylcholine (LysoPC) within serum, or by adding choline as a supplement in serum-free medium [31,32].
In a study of parasite development during the initial ex vivo cycle cultured from clinical samples in Ghana, the proportions of parasites showing conversion to gametocytes correlated positively with level of parasitaemia and negatively with concentration of LysoPC in plasma of patients, supporting a hypothesis that developmental responses to metabolic stress may occur during natural infections [33].
Despite these advances, the quantitative scope of sexual commitment rate variation in P. falciparum has not yet been clearly defined. Estimates of commitment rates have previously varied due to methodological differences or to biological differences between nominally similar parasite lines, and there are few controlled comparisons of different isolates employing biological replicate measurements. Performing multiple independent replicate assays is important, because some individual lines may have temporally variable phenotypes and because precision is generally increased with repeated measurements. In this study, we first compared different methods of measuring sexual commitment rates, and then employed the method with greater quantitative sensitivity and accuracy to conduct multiple biological replicate assays on recently adapted clinical isolates from Ghana as well as a broad selection of long-term laboratory adapted lines representing most of those that are commonly cultured globally. Although each clinical isolate showed variability among repeated assays, overall there are significant differences among isolates, indicating potentially adaptive variation in sexual commitment within this important parasite species.
Separate assays of selected lines on the suppressive effect on sexual commitment by adding choline to serum-free culture media identified significant quantitative variation among lines, which further encourages research to test for relevance to disease transmission.

Comparison of sensitivity and precision of two different assays to measure gametocyte conversion rates
Two different assay methods were compared to quantify sexual commitment rates in multiple independent experiments, using a genetically engineered P. falciparum clone (3D7/iGP_D9) to over-express GDV1 with a destabilising domain (DD) in the presence of the stabilising reagent Shield-1 which leads to elevated gametocyte conversion [34]. Method 1 involved differential counting of stage I gametocytes detected by anti-Pfs16 immunofluorescence staining as a proportion of all parasites developing beyond ring stages in the next cycle, while Method 2 involved Giemsa-stained slide microscopy to compare the next cycle ring stage parasitaemia (referred to as day 0 in this assay, D0) with the parasitaemia of morphologically-defined stage II gametocytes (at day 4, D4) (details given in the Materials and Methods section). In each of multiple biological replicate experiments, both methods clearly show that the gametocyte conversion rate is very significantly higher when GDV1 is overexpressed and stabilised with Shield-1 (GDV1-DD-ON) compared to when  Table S1).
In all biological replicate assays, Method 1 showed a marked difference in gametocyte conversion rate between the GDV1-DD ON and OFF conditions, with a mean conversion rate of 36.9 % under ON and 6.7 % under OFF conditions ( Figure 1). In comparison, Method 2 yielded an estimated mean conversion rate of 15.9 % under ON and 4.1 % under OFF conditions, with the rates for Method 2 being lower than for Method 1 in all replicates for which both were tested in parallel. The precision of the estimates for each of the biological replicates was higher for Method 1, with relatively narrow confidence intervals for each experimental replicate as this method is based on direct counts of developmentally discrete parasites. In comparison, Method 2 yielded wider confidence intervals as it is based on a ratio-of-ratio proportion of two temporally sequential counts of parasitaemia which involves more statistical sampling error (Supplementary Table S1), and this method also depends on successful parasite development over a longer time in culture.

Correlation between gametocyte conversion rate estimates from different assay methods
To test for overall correlation between the estimates from the two assay methods, analysis was performed on data from a broader range of paired assays. In addition to the eight measurements from paired assays performed in the first comparison ( Figure 1) nine additional measurements of the 3D7/iGP_D9 clone were performed with different concentrations of the Shield-1 reagent, to yield a total of 17 paired measurements with both methods for analysis (Supplementary Table S2). Overall, this showed a high and significant nonparametric rank correlation between the results from each assay method (Spearman's rho = 0.70, P = 0.003), with Method 1 generally giving higher conversion rate estimates ( Figure 2) with narrower 95% confidence intervals (Supplementary Table S2). While this supports both assays as separately giving informative relative estimates of gametocyte conversion, Method 1 was chosen for subsequent quantitative analyses of additional lines and culture conditions given its significantly higher sensitivity and tighter precision (Figures 1 and 2, Supplementary Tables S1 and S2).

Variation in sexual commitment rates of recently culture-established Ghanaian clinical isolates
Recent culture-established clinical isolates from a highly endemic area in Ghana were assayed to measure gametocyte conversion rates. Six isolates were selected from a larger panel for analysis as they represent unrelated genetically unmixed infections [35], so that biological replicate assays correspond to a single parasite genotype in each case, and after culture for several months they did not have any known loss of function mutations detected in their genome sequences that would affect sexual commitment [35]. These isolates show mean gametocyte conversion rates that range from 3.3% to 12.2% (Figure 3, Supplementary  Table S4).
There was also significant variation in gametocyte conversion rates among individual biological replicate assays of each isolate, with non-overlapping 95% confidence intervals of some replicates ( Figure 3A). The variation among biological replicate assays for each line was not correlated with the culture parasitaemia, which was always between 0.5 and 2.5% ( Figure 3B and Supplementary Table S3). Overall, there was no significant evidence of reduction in gametocyte conversion rates over time in culture (Supplementary Table S3), and variation among replicates did not correspond with the use of different erythrocyte batches for culture (Supplementary Table S3).

Range of gametocyte conversion rates among diverse long-term cultured laboratory lines
To compare with these recent clinical isolates, a panel of 13 long-term culture-adapted P.
falciparum lines of diverse origins was tested, with multiple biological replicate assays  Table S5). The clone F12 (containing a nonsense mutation in ap2g) [6] here showed zero conversion in all replicate assays ( Figure 4A, Supplementary Table   S5). Four of the other lines showed consistently low levels of gametocyte conversion, with means ranging between 0.3% and 1.6%, significantly lower than the conversion rates of the remaining lines (Mann-Whitney tests on pairwise comparisons, Supplementary Table S6).
The lines with the lowest conversion rates (D10 and T9/96) were previously described as having multiple gene deletions in a sub-telomeric region of chromosome 9 including the gdv1 gene [36]. The 3D7 line may contain undetected mutants within bulk culture, as 3D7 has been elsewhere shown to be a source of parasite subclones with loss of function mutations (including the ap2-g nonsense mutation in parasite subclone F12 [6], deletion of gdv1 in subclone 3D7.Gdef [12] and truncation of gdv1 in two other subclones [37]). The Palo Alto line was previously shown to have undergone sub-telomeric gene deletions on different chromosomes [36,38] but not to have lost the gdv1 locus on chromosome 9 [36,39], although the possibility of a loss-of-function mutation has not been excluded.
For the remaining eight long-term culture-adapted P. falciparum lines, the gametocyte conversion rates for each showed means ranging from 4.7% (for D6) to 14.5% (for FCC2) ( Figure 4A). This range is similar to that seen for the recently culture-established clinical isolates, with no significant differences between these groups (Mann-Whitney test, P = 0.47). Similar to the clinical isolates, the individual long-term culture-adapted parasite lines also showed significant variation in gametocyte conversion rates among biological replicate assays ( Figure 4A), with non-overlapping 95% confidence intervals for some replicates ( Figure 4B). This shows that high and variable sexual commitment rates have been maintained in a majority of the diverse cultured P. falciparum lines, in contrast to a minority of lines that have markedly reduced rates or zero commitment.
Choline in culture medium suppresses sexual commitment rate with varying levels of effect among P. falciparum lines As it has been previously shown that parasite sexual commitment responds to the presence or absence of choline in culture medium [13,28,31], we investigated five long-term culture-  Table S7). No significant effect was seen in the P. falciparum clone T9/96 which has a deletion of gdv1 and several adjacent genes deleted on Chromosome 9 [36], and which has a very low rate of sexual commitment either in the presence or absence of choline, consistent with the importance of gdv1 in responding to environmental induction [13]. The responses of the other lines to choline varied, with statistically significant suppression in sexual commitment in most of the replicate assays for NF54 and Dd2, in half of the assays for HB3, and in a quarter of the assays for 3D7 ( Figure 5). Some individual assay replicates showed more than 10 fold suppression of sexual commitment rate in the presence of choline, but most showed more moderate rates (Supplementary Table S7

Discussion
Analyses of multiple biological replicate assays are informative to characterise the range of sexual commitment rates in P. falciparum. A controlled comparison of two alternative methods showed that phenotyping was more sensitive and precise with an assay that measures proportions of sexually differentiating parasites by immunofluorescence staining of the Pfs16 marker expressed in early stage I gametocytes [15]. Although the sensitivity and precision of the methods differ, they give correlated measurements, and the lower proportions measured using a method that does not employ Pfs16 staining may be expected, given the requirement for parasites to successfully develop to stage II gametocytes over four days in culture before they are counted by morphological inspection of Giemsa-stained slides [33].
The recently culture-established Ghanaian clinical isolates analysed here had gametocyte conversion rates of between 3.3% and 12.2% per cycle, measured as means of results from at least six biological replicate assays for each isolate. These rates are higher than those previously estimated for clinical isolates in the first cycle ex vivo in Ghana with an assay which involves counting of stage II gametocytes, which indicated median rates less than 1% and only approximately a quarter of isolates having rates above 3% [33]. The lower ex vivo rates may be partly due to lower assay sensitivity compared to the Pfs16 staining method, as well as effects of plasma components such as variable lysophosphatidylcholine levels in some individuals inhibiting parasite sexual commitment [33]. It is notable that the isolates in the present study were cultured consistently in serum-free medium supplemented by Albumax-II for several months, demonstrating that use of serum in culture is not required to preserve the intrinsic capacity of parasites to show robust rates of sexual commitment over this period. Nevertheless, parasites with very high rates of sexual commitment might have been selected against, while eventual loss of sexual commitment in some cultures may occur over longer periods of culture.
Most of the diverse long-term laboratory-adapted lines had mean gametocyte conversion rates between 4.7% and 14.5% per cycle, and showed variation in conversion rate among different biological replicates. The only line that always had zero sexual conversion was F12 with a previously described non-functional ap2-g gene due to a nonsense mutation [6]. All other lines showed some detectable sexual conversion, although four lines had much lower rates (means of 0.3 to 1.6%) than the rest, likely due to loss-of-function mutations acquired in culture including deletion of the gdv1 gene in lines T9/96 and D10 [36] which showed the lowest conversion rates. Loss of gdv1 in two other laboratory lines was reportedly associated with no detectable gametocytes [12,37], and it is possible that the low level of conversion in T9/96 and D10 reflects a small amount of commitment to gametocyte development post-invasion due to stochastic activation of ap2-g, independent of the main pathway involving gdv1 [15].
The multiple replicate measurements of each P. falciparum line enable the identification of significant variation among recently derived clinical isolates from the same local endemic population, as well as among long-term laboratory-adapted lines that have diverse geographical origins. Given that modelling of data from experimental infections has indicated much lower rates of approximately 1% or less per cycle [20,21], there may be a significant loss in vivo during the long period of development to circulating late stage gametocytes, or suppression of gametocyte conversion in these infections compared to culture.
Variation in gametocyte conversion rates among biological replicate assays was similar for recently established clinical isolates and most of the long-term laboratory adapted lines.
Measured differences among replicates may reflect intrinsic parasite variation, stochastic or due to determinants yet unidentified, or could be due to assay conditions. Previous studies on a several different culture-adapted P. falciparum clones did not show as much variation between experimental replicates [6,13,28,31], which may be due to tighter parasite synchronisation in those studies, whereas a broad window of synchronisation in the present study was intended to be similar to what may be sampled in peripheral blood (in which ring stage parasites may range in ages up to approximately 24 hours). The inter-replicate assay variation in this study might be partly due to the possibility that a proportion of parasites in some replicates were too young to stain with the Pf16 marker, although this was aimed to be minimised by inspecting parasite development to determine the exact time of assay completion. Although most parasites assayed would be mature enough for Pfs16 staining, some replicates may have parasites at least 36 hours post invasion when all stage I gametocytes would be Pfs16-positive [15,17,40], while other replicates may have parasites 24 hours post invasion when a majority but not all stage I gametocytes may already be Pfs16-positive [15]. Exact standardisation of parasite ages in culture of diverse isolates is not feasible, as isolates intrinsically vary in developmental cycle times [41,42], but it is likely that tighter synchronisation of parasite cultures would reduce variation among replicates for any individual isolate. Assay precision may also be increased if detection assays are developed to enable antibody staining of sexually committed parasites before they express Pfs16, which may become possible as particular gene transcripts are expressed in committed ring stages [43], and at least one protein (GEXP02) is expressed earlier than Pfs16 [17].
Although a majority of the diverse laboratory-adapted parasite lines tested in this study retain an intact mechanism for gametocyte production, there were differences in responses to choline. The presence or absence of choline had no effect in any of eight replicate experiments on the T9/96 clone that has a culture-acquired deletion of gdv1, consistent with the requirement of this locus for mediating a response to induce enhanced levels of sexual commitment [12,13]. In contrast, choline had a significant but variable effect on sexual commitment in each of the other parasite lines tested, each of which also showed substantial variation in responsiveness among biological replicates. Although a few individual replicate assays showed greater than 10 fold effects, mean suppressive effects of choline in each parasite line ranged from 1.5 fold (for 3D7) to 3.5 fold (for Dd2), more moderate than described in previous studies of parasite clones maintained in medium containing choline or serum before being switched to choline-free medium [13,17,31].
Gametocyte conversion rates of >30% have occasionally been reported elsewhere [28], which were not seen in the non-genetically engineered lines here, but only with overexpression of GDV1 in an engineered line. As all parasite lines including clinical isolates in this study were maintained in a standard medium with Albumax without choline supplementation, it is possible that higher gametocyte conversion rates were selected against, as they might not be sustained in continuous culture without using conditions to suppress conversion during long-term maintenance [31].
The results support the utility of choline in medium to suppress parasite sexual commitment under controlled environmental conditions (10,20,21), and notably show significant variation in the responsiveness of different parasite lines. It will be important to understand if there is intraspecific variation in natural regulation of GDV1 expression, particularly the control of gene-silencing antisense transcripts [13], and investigate the potential influence of naturally-occurring cis-variation at the sub-telomeric locus on chromosome 9 [33,39,44] distinct from the deletions disrupting function in some cultured lines [12,36,37,45]. An important new insight into the effect of available choline has just been reported, indicating that sexual commitment is affected by competition between histone methyltransferases (involved in ap2-g gene silencing) and phosphoethanolamine methyltransferase (involved in de novo synthesis of phosphatidylcholine when choline availability is low) for the methyl donor S-adenosylmethionine, which could have implications for future studies on interisolate variation in rates of sexual commitment [32]. Investigation of parasites in natural populations requires assay precision and experimental replication to be considered where possible alongside statistical power from increased numbers of clinical samples, whether for candidate gene investigations or larger scale scanning for associations with unlinked genomic variation in parasite populations [46].

Ethics Approval
Approval for sampling of clinical isolates for culture experimental analysis was granted by the Ethics committees of the Ghana Health Service, the Noguchi Memorial Institute for Medical Research at the University of Ghana, the Navrongo Health Research Centre and the London School of Hygiene and Tropical Medicine, as previously described for the collection [35] from which specific isolates were selected in this analysis.

Plasmodium falciparum laboratory-adapted clones and clinical isolates
The genetically modified P. falciparum cloned line 3D7/iGP_D9 was used to compare the sensitivity and precision of gametocyte conversion rate assays, as artificially elevated sexual commitment is selectively inducible in this line by overexpression of GDV1 fused to a destabilization domain (DD) which maintains activity in the presence of Shield-1 reagent [34].
Thirteen different P. falciparum long-term laboratory-adapted lines that have not undergone targeted genetic modification were studied. Each of these was either previously derived by cloning or is a line that has an apparently monoclonal single-genome sequence, and a putative origin based on the geographical location of the original isolation: NF54 (a strain of 'airport malaria' from The Netherlands with genome sequence similarity to African isolates), 3D7 (the P. falciparum reference genome strain originally cloned from NF54 and maintained in culture for many years) [47], F12 (derived by cloning from cultured 3D7 and having a nonsense mutant ap2-g gene) [6], D10 (Papua New Guinea), D6 (Sierra Leone), Dd2 (Southeast Asia), FCC2 (China), HB3 (Honduras), R033 (Ghana), Palo Alto (Uganda), 7G8 (Brazil), GB4 (Ghana) and T9/96 (Thailand). All lines were thawed from pre-existing frozen stocks with distinct identities verified by previous targeted genotyping and sequencing [39,48] and were not re-cloned prior to this study, and each line was cultured for at least one week after thawing prior to any assay, all culture being conducted using a commerciallyprepared serum-free medium supplemented with lipid-rich bovine serum albumen (Gibco RPMI with 0.5% AlbuMAX™ II lot number 2177881). This consistent medium preparation had very minimal choline (less than 0.02 mM choline chloride) which would not suppress parasite sexual commitment, unlike high concentrations of choline (2 mM as used for some assays described in a separate section below) or serum which has variable components between batches.
Six P. falciparum clinical isolates from Ghana with single-genome unmixed infections were selected from a larger panel of infections sampled from children with clinical malaria in Navrongo (in the Upper East Region of the country), in which parasite asexual multiplication rate variation was previously studied [35]. These isolates were cultured with the same batch of commercially-prepared serum-free medium with 0.5% AlbuMAX™ II as for the laboratoryadapted parasite lines, ensuring the same batch was used throughout all experiments so that no biological variation should be due to media components, and these clinical isolates were not cultured with added serum at any time as they had been cultured with 0.5% AlbuMAX™ II since isolation [35]. Clinical isolates with mixed genome-infections were purposefully not selected for inclusion in this study, as their sexual commitment rates would be complex to interpret due to potential changes in mixed genomic composition over time [35].

Quantification of sexual commitment rates by measuring gametocyte conversion
Multiple biological replicate preparations of P. falciparum long-term laboratory-adapted lines and more recently culture-established clinical isolates were each cultured in human erythrocytes at 3% hematocrit in RPMI 1640 media supplemented with AlbuMAX™ II and incubated at 37°C in atmospheric air with 5% CO2. Prior to assay, each asynchronous parasite culture was synchronized to within a 24-hour window of intra-erythrocytic asexual stage development, intended to correspond approximately to the parasite age range that would be typically sampled in peripheral blood of patients, using the following two-step technique. Parasites containing haemozoin, predominantly schizont and late trophozoite stages, were first positively selected on MACS® LD magnetic columns [49], and after culture for 24 hours a second MACS purification step was performed from which the flow through erythrocytes (containing predominantly ring stage parasites) were collected and put back into culture, which then contained exclusively ring stage parasites as any gametocytes or residual mature asexual parasites were removed on the column. This broad synchronization scheme is illustrated in Supplementary Figure S1 Two methods were used for measuring the gametocyte conversion rate:

Method 1. Counting of the proportion of stage I gametocytes compared to all parasites developing in the first cycle post-invasion using anti-Pfs16 staining.
This method involves distinguishing very early gametocytes from asexual parasites by detection of the gametocyte marker Pfs16 protein [15,40,50]. After two rounds of MACS column purification to obtain ring stages followed by a further 24 hours of growth, when a culture with a broad synchronicity containing schizonts was obtained, parasites were allowed to invade erythrocytes and were cultured for a period of between 38 and 46 hours, to the previous cycle were not counted). The assay enables most sexually-developing parasites to be counted in comparison to the total of post-invasion parasites, on the basis that a majority of early stage I gametocytes express Pfs16 after 24 hours post invasion [15] as a proportion of all gametocytes which eventually express [40], with only a minority of sexual parasites likely not to be counted due to being too young. Parasite cultures harvested for this purpose were washed in phosphate buffered saline (PBS) with 3% bovine serum albumen (BSA), resuspended to 2% hematocrit, spotted onto multiwell slides (Hendley, Essex, UK), air-dried and stored at -80°C. Prior to immunofluorescence staining, slides were fixed with 4% paraformaldehyde in PBS for 30 min and permeabilized with 0.1% Triton X-100 in PBS for 10 min. The slides were incubated for 30 minutes at room temperature with a monoclonal α-Pfs16 murine antibody 93A3A2 [50], diluted 1:2000 in PBS with 3% BSA. Alexa Fluor® 594-conjugated anti-mouse IgG (Invitrogen, A11032) was used as secondary antibody, diluted 1:1000 in PBS with 3% BSA and incubated for 30 minutes at room temperature. Slides were mounted with coverslips using DAPI-containing Prolong TM Diamond antifade mountant (ThermoFisher Scientific), and parasite counting was performed using a Zeiss CCD fluorescence microscope, with identical settings used for each experiment.
An average of approximately 1000 parasites were counted to determine proportions expressing Pfs16 in each measurement, and where the density of parasites did not allow this a quality control was performed to exclude any assay data that did not have a minimum of at least 300 parasites counted. This method estimates the proportion of parasites that produce gametocytes developing to morphological stage II, by comparing with the ring parasitaemia counted 4 days previously at the beginning of the parasite developmental cycle. The technique is based on a protocol that uses Giemsa staining and does not require immunofluorescence staining of an early gametocyte marker [33]. To initiate this assay, after two MACS column purifications and when a broadly synchronous culture containing a majority of schizonts were obtained, parasites were allowed to re-invade fresh erythrocytes and cultured until ring stage development was observed. Then, ring stage parasitaemia was assessed by visual examination of thin blood smears of each culture to determine the proportion of erythrocytes containing parasites (referred to as D0 parasitaemia), ensuring that at least 1,000 erythrocytes were counted. The cultures were then treated with 50 mM N-acetyl-Dglucosamine (GlcNAc) to prevent further development of asexual parasites [51]. After 4 days, the stage II gametocytemia was assessed by counting of Giemsa-stained thin blood smears (referred to as D4 gametocytemia), ensuring that at least 10,000 erythrocytes were counted. The gametocyte conversion rate was calculated by dividing D4 stage II gametocytemia (as a proportion of erythrocytes infected) by D0 ring parasitaemia (as a proportion of erythrocytes infected), yielding a 'ratio-of-ratio' proportion with 95% confidence intervals.
For both methods, all assays were set up at 3% haematocrit in a culture volume of 5 ml (static in 6-well culture plates), with parasitaemia levels at the start of each replicate assay being between 0.5 -2.5% erythrocytes infected, to allow sufficient numbers of parasites for high precision counting while minimising stress on parasites that may be induced if cultures have higher parasitaemia.

Quantification of gametocyte conversion rate using culture media without choline
Parasites were prepared for assays in the same manner as described in the previous section, except that they were split after the second MACS column purification (at ring stage in the cycle before gametocyte counting) into separate wells with culture media (RPMI 1640 with 25 mM HEPES, 100 µM Hypoxanthine, 1 mM L-Glutamine, 0.39% fatty acid-free BSA, 30 mM Palmitic acid, 30 mM Oleic acid) either supplemented with 2 mM choline or lacking choline, based on a previously described protocol [31]. Parasites were allowed to grow until schizont stage and to invade fresh erythrocytes, without changing the culture medium. After 38-46 hours post invasion, parasites were collected and fixed for analysis of gametocyte conversion by differential counting using Pfs16 staining by immunofluorescence microscopy as well as DAPI staining as described above for Method 1, with an average of approximately 1000 parasites being counted for each individual measurement. Between six and eight replicate assays were performed for each parasite line, the effect within each replicate calculated as the rate ratio (with 95% confidence intervals) of the gametocyte conversion rate in choline-free medium compared with the conversion rate in medium containing 2 mM choline.

Statistical analyses
Spearman's ρ (rho) coefficient was used to evaluate the strength and significance of the rank correlation between results from the two different gametocyte conversion rate assays    staining to discriminate sexually from asexually-developing parasites) and Method 2 (D0D4, involving counts of parasites 4 days apart) on multiple biological replicates of 3D7/iGP_D9 with a range of concentrations of Shield-1 reagent (N=17 paired assays in total, details in Supplementary Table S2). This highly significant correlation confirms that either method gives valid measurements if used alone, whereas Method 1 is more sensitive (as indicted here and in Figure 1). Method 1 is also more precise as it involves calculation of a single numerical ratio, rather than a ratio-of-ratios based on two different counts performed 4 days apart which is highly vulnerable to effects of small numbers in either count. For visual clarity, the individual assay 95% confidence intervals are not shown on the plot, but these and numerical data of all assays are given in Supplementary Table S2. isolates. These were selected for study as they are unrelated single genotype infections, chosen from a larger number of samples from a highly endemic population that contained many multiple genotype infections [35]. A. For each isolate, a minimum of six biological replicate assays was performed, and the estimate from each replicate with 95% confidence intervals (based on numbers of parasites counted) is plotted, while the red dotted line represents the mean across replicate assays. There were significant differences in pairwise comparisons (Mann-Whitney tests, Supplementary  Table S4). There were no significant correlations with length of time in culture, or batches of erythrocytes used for the different replicate assays (these details and all numerical data values are given in Supplementary Table S3). B. For each isolate the gametocyte conversion rate of individual replicates is plotted in relation to the culture parasitaemia in the previous cycle (between 0.5 and 2.5% for all experimental replicates). 95% confidence intervals individual conversion rate points are not shown as they are the same as in panel A. There was no systematic trend towards positive or negative correlation, except a marginally significant positive correlation for isolate 289 (P = 0.03) which may indicate slight enhancement at higher parasitaemia or a chance finding due to multiple comparisons.  Table S5).