Evolutionary sex allocation theory explains sex ratios in natural Plasmodium falciparum infections

Malaria transmission is achieved by sexual stages, called gametocytes, and the proportion of gametocytes that are male versus female (sex ratio) influences transmission success. In malaria model systems, variation in gametocyte sex ratios can be explained by the predictions of evolutionary sex allocation theory. We test these predictions using natural Plasmodium falciparum infections. The predicted negative correlation between sex ratio and gametocyte density holds: the sex ratio increases when gametocyte densities decrease, and this is most apparent in single genotype infections and in the dry season. We do not observe higher gametocyte sex ratios in mixed compared with single genotype infections.     

Density-dependent impact of the human malaria parasite Plasmodium falciparum gametocyte sex ratio on mosquito infection rates

Malaria parasites produce male and female life cycle stages (gametocytes) that must fertilize to achieve successful colonization of the mosquito. Gametocyte sex ratios have been shown to be under strong selection pressure both as an adaptive response to a worsening blood environment for transmission and according to the number of co-infecting clones in the vertebrate. Evidence for an impact of sex ratio on the transmission success of Plasmodium falciparum has, however, been more controversial. Theoretical models of fertilization predict that increasingly male sex ratios will be favoured at low gametocyte densities to ensure fertilization. Here, we analyse in vitro transmission studies of P. falciparum to Anopheles gambiae mosquitoes and test this prediction. We find that there is a discernible effect of sex ratio on transmission but which is dependent upon the gametocyte density. While increasingly male sex ratios do give higher transmission success at low gametocyte densities, they reduce success at higher densities. This therefore provides empirical confirmation that sex ratio has an immediate impact on transmission success and that it is density-dependent. Identifying the signals used by the parasite to alter its sex ratio is essential to determine the success of transmission-blocking vaccines that aim to impede the fertilization process.     

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

The effects of artemisinin-based combination therapies (ACTs) on transmission of Plasmodium falciparum were evaluated after a policy change instituting the use of ACTs in an endemic area. P. falciparum gametocyte carriage, sex ratios and inbreeding rates were examined in 2,585 children at presentation with acute falciparum malaria during a 10-year period from 2001-2010. Asexual parasite rates were also evaluated from 2003-2010 in 10,615 children before and after the policy change. Gametocyte carriage declined significantly from 12.4% in 2001 to 3.6% in 2010 (χ2 for trend = 44.3, p < 0.0001), but sex ratios and inbreeding rates remained unchanged. Additionally, overall parasite rates remained unchanged before and after the policy change (47.2% vs. 45.4%), but these rates declined significantly from 2003-2010 (χ2 for trend 35.4, p < 0.0001). Chloroquine (CQ) and artemether-lumefantrine (AL) were used as prototype drugs before and after the policy change, respectively. AL significantly shortened the duration of male gametocyte carriage in individual patients after treatment began compared with CQ (log rank statistic = 7.92, p = 0.005). ACTs reduced the rate of gametocyte carriage in children with acute falciparum infections at presentation and shortened the duration of male gametocyte carriage after treatment. However, parasite population sex ratios, inbreeding rates and overall parasite rate were unaffected.     

Changes in Haemoproteus sex ratios: fertility insurance or differential sex lifespan?

There is little direct evidence of the fitness effects of changes in malaria gametocyte sex ratio. Gametocyte sex ratios in haemospororin parasites (phylum Apicomplexa) are usually female skewed. However, in some cases and especially in Haemoproteus parasites, less female-biased and even male-biased sex ratios are encountered. The 'fertility insurance hypothesis' tries to explain these biases as an evolutionary strategy to facilitate gamete encounter. Thus, the hypothesis predicts that, if there is a reduction in gametocyte density (intensity of infection) or other factors preventing gametes from meeting, a change to a higher proportion of male gametocytes may be favoured. By contrast, a change in sex ratio may be caused by other non-adaptive mechanisms, for example differential survival of the gametocytes of each sex. We study within-host changes in Haemoproteus majoris sex ratios following an experimental reduction in the density of the parasites in the blood in a breeding population of blue tits (Parus caeruleus). Medication with the antimalarial drug primaquine induced a significant reduction in Haemoproteus gametocyte infection intensity in two different breeding seasons and under two different doses of medication. Sex ratios became male skewed following the experimental treatment in agreement with the predictions of the 'fertility insurance' hypothesis. Also in support of the hypothesis, a significant change towards male-biased sex ratios emerged for non-medicated birds in one year, probably owing to the natural immune reduction of the density of the parasites in the blood. The alternative possibility that changes are caused by different lifespans of gametocytes is not supported by changes in sex ratios in control hosts, where new production and release of gametocytes occur.     

Gametocyte sex ratio of a malaria parasite: experimental test of heritability

The gametocyte sex ratio of Plasmodium mexicanum, a malaria parasite of western fence lizards, was studied in a modified garden experiment. Each of 6 naturally infected lizards was used to initiate 20 replicate-infections in naive western fence lizards. A significant donor effect was observed for the sex ratios of recipient infections at their maximal parasitemia, and this effect was associated with the sex ratio of the donor infection. In 20 infections in which sex ratio was followed during the course of the infection, 9 revealed constant sex ratios and 11 showed an increase in proportion of males over time. Recipient sex ratio was correlated with another life-history trait, a composite of rate of asexual replication and peak parasitemia, such that higher Rate-Peak scores were associated with infections with less female-biased sex ratios. These results are placed into the context of sex ratio theory that concludes that the degree of selfing of parasite genotypes (number of parasite clones) within the vector will influence the evolution of gametocyte sex ratio. The theory predicts that the sex ratio should be under some genetic control and thus be heritable as observed in the experiment. Clonal diversity should also influence the life-history trait, Rate-Peak, which was found to be correlated with sex ratio.     

Plasmodium falciparum gametocyte carriage, emergence, clearance and population sex ratios in anaemic and non-anaemic malarious children

Anaemia in falciparum malaria is associated with an increased risk of gametocyte carriage, but its effects on transmission have not been extensively evaluated in malarious children. Plasmodium falciparum gametocyte carriage, emergence, clearance, population sex ratios (SR) (defined as the proportion of gametocytes that are male), inbreeding rates and temporal changes in SR were evaluated in 840 malarious children. Gametocyte carriage pre-treatment was at a level of 8.1%. Anaemia at enrolment was an independent risk factor for gametocyte carriage post-treatment. The emergence of gametocytes seven days post-treatment was significantly more frequent in anaemic children (7/106 vs. 10/696, p = 0.002). In the initially detected gametocytes, the proportion of children with a male-biased SR (MBSR) (> 0.5) was significantly higher in anaemic children (6/7 vs. 3/10, p = 0.027). Pre-treatment SR and estimated inbreeding rates (proportion of a mother's daughters fertilised by her sons) were similar in anaemic and non-anaemic children. Pre-treatment SR became more female-biased in non-anaemic children following treatment. However, in anaemic children, SR became male-biased. Anaemia was shown to significantly increase gametocyte emergence and may significantly alter the SR of emerging gametocytes. If MBSR is more infective to mosquitoes at low gametocytaemia, then these findings may have significant implications for malaria control efforts in endemic settings where malaria-associated anaemia is common.     

Sex allocation and population structure in apicomplexan (protozoa) parasites

Establishing the selfing, rate of parasites is important for studies in clinical and epidemiological medicine as well as evolutionary biology Sex allocation theory offers a relatively cheap and easy way to estimate selfing rates in natural parasite populations. Local mate competition (LMC) theory predicts that the optimal sex ratio (r*; defined as proportion males) is related to the selfing rate (s) by the equation r* = (1-s)/2. In this paper, we generalize the application of sex allocation theory across parasitic protozoa in the phylum Apicomplexa. This cosmopolitan phylum consists entirely of parasites, and includes a number of species of medical and veterinary importance. We suggest that LMC theory should apply to eimeriorin intestinal parasites. As predicted, data from 13 eimeriorin species showed a female-biased sex ratio, with the sex ratios suggesting high levels of selfing (0.8-1.0). Importantly, our estimate of the selfing rate in one of these species, Toxoplasma gondii, is in agreement with previous genetic analyses. In contrast, we predict that LMC theory will not apply to the groups in which syzygy occurs (adeleorins, gregarines and piroplasms). Syzygy occurs when a single male gametocyte and a single female gametocyte pair together physically or in close proximity, just prior to fertilization. As predicted, data from four adeleorin species showed sex ratios not significantly different from 0.5.     

Effects of antifolates--co-trimoxazole and pyrimethamine-sulfadoxine--on gametocytes in children with acute, symptomatic, uncomplicated, Plasmodium falciparum malaria

Antimalarial drugs including the antifolate, pyrimethamine-sulfadoxine (PS), can modulate the prevalence and intensities of gametocytaemia following treatment of acute malaria infections. They may also directly influence the transmission and spread of drug insensitivity. Little is known of the effects of co-trimoxazole (Co-T), another antifolate antimalarial, on gametocytes in children with acute malaria infections. We compared the effects of Co-T and PS on the prevalence and intensities of gametocytaemia and gametocyte sex ratios in 102 children aged 0.5-12 years presenting with acute and uncomplicated falciparum malaria. Compared to pre-treatment, both drugs significantly increased gametocyte carriage post-initiation of treatment. However, gametocyte carriage was significantly lower on day 14 in those treated with Co-T than PS. Significant increase in gametocytaemia with time occurred in PS--but not Co-T-treated children. Kaplan-Meier survival curve of the cumulative probability of remaining gametocyte-free in children who were agametocytaemic at enrollment showed that by day 7 of follow up, children treated with PS had a significantly higher propensity to have developed gametocytes than in Co-T-treated children (Log-rank statistic 5.35, df = 1, P = 0.02). Gametocyte sex ratio changes were similar following treatment with both drugs. PS and Co-T treatment of acute malaria infections in children from this endemic area is associated with significant increases in prevalence and intensities of gametocytaemia but these effects are more marked in those treated with PS than Co-T.     

Manipulation of the vertebrate host's testosterone does not affect gametocyte sex ratio of a malaria parasite

Gametocyte sex ratio of the malaria parasite Plasmodium mexicanum is variable in its host, the western fence lizard (Sceloporus occidentalis), both among infections and within infections over time. We sought to determine the effect of host physiological quality on the gametocyte sex ratio in experimentally induced infections of P. mexicanum. Adult male lizards were assigned to 4 treatment groups: castrated, castrated + testosterone implant, sham implant, and unmanipulated control. No significant difference in gametocyte sex ratio was found among the 4 treatment groups. Two other analyses were performed. A surgery stress analysis compared infection sex ratio of castrated, castrated + testosterone implant, and sham implant groups with the unmanipulated control group. A testosterone alteration analysis compared infection sex ratio of the castrated and castrated + testosterone implant groups with the sham implant and unmanipulated control groups. Again, no significant difference was observed for these 2 comparisons. Thus, physiological changes expected for experimentally induced variation in host testosterone and the stress of surgery were not associated with any change in the gametocyte sex ratio. Also, theex-periment suggests testosterone is not a cue for shaping the sex ratio of gametocytes in P. mexicanum. These results are related to the evolutionary theory of sex ratios as applied to malaria parasites.     

Sex-partitioning of the Plasmodium falciparum Stage V Gametocyte Proteome Provides Insight into falciparum-specific Cell Biology

One of the critical gaps in malaria transmission biology and surveillance is our lack of knowledge about Plasmodium falciparum gametocyte biology, especially sexual dimorphic development and how sex ratios that may influence transmission from the human to the mosquito. Dissecting this process has been hampered by the lack of sex-specific protein markers for the circulating, mature stage V gametocytes. The current evidence suggests a high degree of conservation in gametocyte gene complement across Plasmodium, and therefore presumably for sex-specific genes as well. To better our understanding of gametocyte development and subsequent infectiousness to mosquitoes, we undertook a Systematic Subtractive Bioinformatic analysis (filtering) approach to identify sex-specific P. falciparum NF54 protein markers based on a comparison with the Dd2 strain, which is defective in producing males, and with syntenic male and female proteins from the reanalyzed and updated P. berghei (related rodent malaria parasite) gametocyte proteomes. This produced a short list of 174 male- and 258 female-enriched P. falciparum stage V proteins, some of which appear to be under strong diversifying selection, suggesting ongoing adaptation to mosquito vector species. We generated antibodies against three putative female-specific gametocyte stage V proteins in P. falciparum and confirmed either conserved sex-specificity or the lack of cross-species sex-partitioning. Finally, our study provides not only an additional resource for mass spectrometry-derived evidence for gametocyte proteins but also lays down the foundation for rational screening and development of novel sex-partitioned protein biomarkers and transmission-blocking vaccine candidates.Sexual stages represent only a small fraction of Plasmodium falciparum parasites that are present during human malaria infection, yet they alone are responsible for disease transmission (1). As such, the Malaria Eradication Research Agenda (malERA) has prioritized the need for studies that specifically address these transmission stages, with the hope of developing new transmission-blocking vaccines and drugs, as well as diagnostics that are specific for these sexual stages (2–4). In fact, one of the critical gaps in malaria transmission biology and surveillance centers on the lack of knowledge about the infectivity of symptomatic and asymptomatic gametocytemic individuals for mosquitoes. Many infected individuals harboring the Plasmodium falciparum sexual stage, or gametocyte, are asymptomatic carriers and they represent the primary reservoir for malaria transmission (5). Missing the opportunity to treat these carriers will increase the risk for epidemic malaria in regions that have approached the elimination phase. Thus, proper surveillance of gametocyte carriers is critical for evaluating ongoing malaria control and elimination programs. Surveillance is difficult, however, because gametocytes comprise only 0.1–2% of the total body parasite load during active infection (5), and are only observed in the bloodstream in their mature (Stage V) form, with the first four developing stages sequestered in tissues. Microscopy-based analysis for sex ratio determination and infectivity studies remains limited because of cost, training, and suitability for population-wide studies. Although light microscopy remains the gold standard for malaria diagnosis, the relatively low prevalence of circulating gametocytes makes it difficult to accurately detect much less quantify these stages. Moreover, because of variations in skill level of microscopists and inconsistency in method, exclusive use of light microscopy estimates of gametocyte carriage carries a high risk of error. Importantly, the presence of stage V gametocytes in the bloodstream alone, as determined by thick smear microscopy does not imply infectivity to mosquitoes. Ratios of male and female gametocytes in the blood circulation are skewed toward the female, but they can vary significantly based on co-infection, parasite and gametocyte density, and host environmental factors (6), and it is therefore hypothesized that this variation in sex ratios will influence mosquito infectivity. For example, mature gametocyte sex ratios can change during the course of infection in response to host cues or especially following antimalarial treatment resulting in an increase in the number of males (6, 7). However, it remains unknown whether the transmission potential to mosquitoes of the individuals in these studies fluctuated because of the changes in sex ratio.There are currently no uncomplicated tools to distinguish male and female mature P. falciparum gametocytes (of which at least one of each is required for fertilization and ookinete development in the mosquito) at the molecular level. Although the proteome of Plasmodium gametocytes has been described (8–11), these previous analyses fell just short of providing the partitioned male and female proteomes for P. falciparum. Moreover, the availability of the genomes of human, primate, and rodent malaria parasites and the acquisition of sequence information for recent field isolates of P. falciparum have created the opportunity to understand gene diversity and conservation in sexual stage development across Plasmodia. Identifying markers that differ between male and female P. falciparum stage V gametocytes is critical in informing transgenic approaches aimed at separating the two. It has been argued that the inherent evolutionary differences between rodent and human malaria parasites, especially for the sexual stages, limit the utility of the P. berghei gametocyte proteome (11) in providing a priori knowledge of these markers. Several iterations and improvements to the P. berghei genome have been made available since 2005, whereas MS search engines have improved commensurately, further compounding the issue. However, we would also argue that the current evidence suggests a high degree of conservation in gametocyte gene complement across Plasmodium (12, 13), and therefore presumably in sex-specific genes - despite key differences such as gametocyte sequestration and morphology. Here, we report on our effort to address these scientific gaps to a certain extent and to test our gametocyte gene conservation hypothesis through the use of comparative protein bioinformatics analyses of the mature stage V gametocyte proteomes of two distinct P. falciparum strains with our update of the bioinformatic analysis of the P. berghei male and female gametocyte proteomes.     

Fertility insurance and the sex ratios of malaria and related hemospororin blood parasites

The sex ratio (z*; proportion of gametocytes that are male) of malaria and related hemospororin blood parasites has been predicted to be related to the inbreeding rate (f) by the simple equation z* = (1 - f)/2. Although there is some empirical support for this prediction, there are several cases where the sex ratio is less female biased or more variable than expected. Here, we present a theoretical model that may be able to explain some of these discrepancies. We show that if low gametocyte densities lead to a danger that female gametes may not encounter any male gametes, then natural selection favors a less female-biased sex ratio as a form of 'fertility insurance' to ensure that female gametes are mated. This model can be applied to a number of situations. In particular, (1) empirical data suggest that the number of gametocyes per blood meal can be low enough to favor fertility insurance in some Plasmodium infections in humans and (2) our model predicts facultative shifting toward less-biased sex ratios in response to immune pressure that reduces gametocyte or gamete survival or mobility, consistent with some recent experimental data from Plasmodium species of birds and mice.     

Population structure of Plasmodium falciparum gametocyte sex ratios in malarious children in an endemic area

The gametocyte sex ratio (proportion of gametocytes that are male) of Plasmodium falciparum may influence transmission. The distribution of P. falciparum sex ratios, the extent of inbreeding, the relationship between clone multiplicity and sex ratio, and the pre- and post-treatment factors influencing a sex ratio of 0.5 were determined in 1609 children, with acute malaria. Gametocytes were sexed by morphological appearance and asexual clone multiplicity was determined by polymerase chain reaction (PCR) using polymorphic loci of merozoite surface proteins-1 and -2 (MSP-1, MSP-2) and glutamine-rich protein (GLURP). The weighted mean population sex ratio on presentation in 162 gametocyte carriers was 0.22, that is, 3.5 female to 1 male (95% CI 0.15–0.28), with an estimated inbreeding rate (f) (the proportion of a mother's daughters that is fertilized by her sons) of 0.56 (95% CI 0.44–0.70). Sex ratio was significantly higher when clone multiplicity was >1 infecting clone than when it was 1 (P = 0.02). The frequency of a pre-treatment sex ratio of 0.5 was low (3%), and was significantly increased by non-artemisinin but not by artemisinin – mono or combination – drugs by day 7 after therapy commenced (P = 0.03 and P = 0.44, respectively). No factor was associated with a pre-treatment sex ratio of 0.5 but two factors were independent predictors of a sex ratio of 0.5 by day 7 after therapy commenced: an age ≥5 years and anaemia. These population data provide some empirical support for the predictions of local mate competition (LMC) theory and, in conjunction with effects of antimalarials on a sex ratio of 0.5, may have implications for malaria control efforts in endemic settings.     

Risk Factors for Plasmodium falciparum Gametocyte Positivity in a Longitudinal Cohort

Malaria transmission intensity is highly heterogeneous even at a very small scale. Implementing targeted intervention in malaria transmission hotspots offers the potential to reduce the burden of disease both locally and in adjacent areas. Transmission of malaria parasites from man to mosquito requires the production of gametocyte stage parasites. Cluster analysis of a 19-year long cohort study for gametocyte carriage revealed spatially defined gametocyte hotspots that occurred during the time when chloroquine was the drug used for clinical case treatment. In addition to known risk factors for gametocyte carriage, notably young age (<15 years old) and associated with a clinical episode, blood groups B and O increased risk compared to groups A and AB. A hotspot of clinical P. falciparum clinical episodes that overlapped the gametocyte hotspots was also identified. Gametocyte positivity was found to be increased in individuals who had been treated with chloroquine, as opposed to other drug treatment regimens, for a clinical P. falciparum episode up to 30 days previously. It seems likely the hotspots were generated by a vicious circle of ineffective treatment of clinical cases and concomitant gametocyte production in a sub-population characterized by an increased prevalence of all the identified risk factors. While rapid access to treatment with an effective anti-malarial can reduce the duration of gametocyte carriage and onward parasite transmission, localised hotspots represent a challenge to malaria control and eventual eradication.     

Toxoplasma gondii,sex and premature rejection

Adaptive sex ratio theory explains why gametocyte sex ratios are female-biased in many populations of apicomplexan parasites such as Plasmodium and Toxoplasma. Recently, Ferguson has criticized this framework and proposed two alternative explanations--one for vector-borne parasites (e.g. Plasmodium) and one for Toxoplasma. Ferguson raises some interesting issues that certainly deserve more empirical attention. However, it should be pointed out that: (1) there are theoretical and empirical problems for his alternative hypotheses; and (2) existing empirical data support the application of sex ratio theory to these parasites, not its rejection.     

TESTING SEX RATIO THEORY WITH THE MALARIA PARASITE PLASMODIUM MEXICANUM IN NATURAL AND EXPERIMENTAL INFECTIONS

The malaria parasite (Plasmodium) life history accords well with the assumptions of local mate competition (LMC) of sex ratio theory. Within a single meal of the blood‐feeding vector, sexually dimorphic gametocyte cells produce gametes (females produce one, males several) that mate and undergo sexual recombination. The theory posits several factors drive the Plasmodium sex ratio: male fecundity (gametes/male gametocyte), number and relative abundance of parasite clones, and gametocyte density. We measured these traits for the lizard malaria parasite, Plasmodium mexicanum, with a large sample of natural infections and infections from experiments that manipulated clonal diversity. Sex ratio in single‐clone infections was slightly female‐biased, but matched predictions of theory for this low‐fecundity species. Sex ratio was less female‐biased in clonally diverse infections as predicted by LMC for the experimental, but not natural infections. Gametocyte density was not positively related to sex ratio. These results are explained by the P. mexicanum life history of naturally low clonal diversity and high gametocyte production. This is the first study of a natural malaria system that examines all traits relevant to LMC in individual vertebrate hosts and suggests a striking example of sex ratio theory having significance for human public health.     

The effect of partial host immunity on the transmission of malaria parasites.

Experiments were carried out to determine the effect of partial host immunity against the rodent malaria parasite Plasmodium chabaudi on the transmission success of the parasite. There was a fourfold reduction in both the blood-stage, asexually replicating parasite density and the gametocyte (transmissable stage) density in immunized hosts. Some of the reduction in asexual parasite densities was due to strain-specific immunity, but there was no evidence that strain-specific immunity affected gametocyte densities. However, immunity did affect transmission in a strain-specific manner, with a fivefold reduction in gametocyte infectivity to mosquitoes in homologous challenges compared with heterologous challenges or non-immunized controls. This implies the existence of a mechanism of strain-specific infectivity-reducing immunity that does not affect the density of gametocytes circulating in peripheral blood. The proportion of asexual parasites that produced gametocytes increased during the course of infection in both non-immunized and in immunized hosts, but immunity increased gametocyte production early in the infection.     

Even more extreme fertility insurance and the sex ratios of protozoan blood parasites

Theory developed for malaria and other protozoan parasites predicts that the evolutionarily stable gametocyte sex ratio (z*; proportion of gametocytes that are male) should be related to the inbreeding rate (f) by the equation z*=(1-f)/2. Although this equation has been applied with some success, it has been suggested that in some cases a less female biased sex ratio can be favoured to ensure female gametes are fertilized. Such fertility insurance can arise in response to two factors: (i) low numbers of gametes produced per gametocyte and (ii) the gametes of only a limited number of gametocytes being able to interact. However, previous theoretical studies have considered the influence of these two forms of fertility insurance separately. We use a stochastic analytical model to address this problem, and examine the consequences of when these two types of fertility insurance are allowed to occur simultaneously. Our results show that interactions between the two types of fertility insurance reduce the extent of female bias predicted in the sex ratio, suggesting that fertility insurance may be more important than has previously been assumed.