Malaria is the major cause of mortality and morbidity in tropical countries. The causative agent, Plasmodium sp., has a complex life cycle and is armed with various mechanisms which ensure its continuous transmission. Gametocytes represent the sexual stage of the parasite and are indispensable for the transmission of the parasite from the human host to the mosquito. Despite its vital role in the parasite's success, it is the least understood stage in the parasite's life cycle. The presence of gametocytes in asymptomatic populations and induction of gametocytogenesis by most antimalarial drugs warrants further investigation into its biology. With a renewed focus on malaria elimination and advent of modern technology available to biologists today, the field of gametocyte biology has developed swiftly, providing crucial insights into the molecular mechanisms driving sexual commitment. This review will summarise key current findings in the field of gametocyte biology and address the associated challenges faced in malaria detection, control and elimination.

The highly pathogenic avian protozoan Leucocytozoon caulleryi infects host chicken cells, and interference by the host genome results in difficulty in obtaining protozoal DNA for genetic analysis. We used flow cytometry analysis to separate expelled L. caulleryi gametocytes from infected chicken blood and to analyse cell populations and sorting by FACS efficiency. Infected blood cells stained with SYTO-24 showed a specific area on 2-dimensional scattergrams compared to uninfected blood. The specific area was sorted, and approximately 85% of the sorted cells were identified as L. caulleryi gametocytes by microscopic observation. DNA was also extracted from the sorted fraction, and a clear increase in polymerase chain reaction (PCR) amplification of protozoal DNA was observed compared to infected blood without sorting. Host-derived DNA was also detected by PCR; however, its amplification was decreased compared to that in unsorted infected blood. This is the first report of the separation of L. caulleryi gametocytes from infected host blood using flow cytometry. This method may be applied to further genetic analyses such as studies of the dynamics of stage-specific L. caulleryi gene expression.

Sex ratio theory predicts that malaria parasites should bias gametocyte production toward female cells in single-clone infections because they will experience complete inbreeding of parasite gametes within the vector. A higher proportion of male gametocytes is favoured under conditions that reduce success of male gametes at reaching females such as low gametocyte density or attack of the immune system later in the infection. Recent experimental studies reveal genetic variation for gametocyte sex ratio in single-clone infections. We examined these issues with a study of experimental single-clone infections for the lizard malaria parasite Plasmodium mexicanum in its natural host. Gametocyte sex ratios of replicate single-clone infections were determined over a period of 3–4 months. Sex ratios were generally female biased, but not as strongly as expected under simple sex ratio theory. Gametocyte density was not related to sex ratio, and male gametocytes did not become more common later in infections. The apparent surplus of male gametocytes could be explained if male fecundity is low in this parasite, or if rapid clotting of the lizard blood reduces male gamete mobility. There was also a significant clone effect on sex ratio, suggesting genetic variation for some life-history trait, possibly male fecundity.

Using cloned lines of Plasmodium berghei producing mixed asexual and sexual (clone 234L) and purely asexual (clone 233L) parasitaemias, the courses of parasitaemia, gametocytogenesis, exflagellation, ookinete production in vitro and mosquito infectivity have been followed. For clone 234L mosquito infectivity is maximal at day 3 and has ceased by day 6 post-infection. Conversely, gametocytogenesis, exflagellation and ookinete production are at minimal levels at day 3 and rise to peaks between days 10 and 15 of infection (in TO mice infected with blood at mechanical blood passage 3). Sexual potential declines progressively with sustained mechanical passage (up to P14). Gametocyte conversion is highest early in infection and declines exponentially; however, upon each mechanical passage conversion is again raised but decreases more rapidly with succeeding passages. For practical mosquito transmission in the laboratory we consider P8 to be the useful limit for mechanical transmission for this parasite clone. Asexual parasite growth (virulence) is more rapid with increased mechanical passage inducing a rapid fall in haematocrit. By P14 the course of infection closely parallels that of the purely asexual clone 233L.

In the rodent malarial parasite Plasmodium berghei sexual parasites are produced in a single major wave with maximal numbers between day 7 and day 16. Irrespective of their time of appearance during infection these sexual parasites are equally fertile in vitro. In contrast, in vivo infectivity to the mosquito is maximal at day 3–5 when gametocyte numbers are only 9% of the peak levels seen between days 7 and 16. Up to 96% of natural potential infectivity of gametocytes for the mosquito is therefore suppressed. The suppression is humoral, reversible and correlates with the appearance of an effective host response to the initial rapid increase in asexual parasitaemia. These data are consistent with published evidence which indicates that a reduction in parasitaemia may cause an increase in infectivity of gametocytes to the mosquito vector. Therefore the impact of strategies aiming to control asexual parasites is re-examined. Inefficient strategies might be predicted to increase and not suppress malaria transmission.

The sexual phase of the malaria pathogen, Plasmodium falciparum, culminates in fertilization within the midgut of the mosquito and represents a crucial step in the completion of the parasite's life-cycle and transmission of the disease. Two decades ago, the first sexual stage-specific surface proteins were identified, among them Pfs230, Pfs48/45, and Pfs25, which were of scientific interest as candidates for the development of transmission blocking vaccines. A decade later, gene information gained from the sequencing of the P. falciparum genome led to the identification of numerous additional sexual-stage proteins with antigenic properties and novel enzymes that putatively possess regulatory functions during sexual-stage development. This review aims to summarize the sexual-stage proteins identified to date, to compare their stage specificities and expression patterns and to highlight novel regulative mechanisms of sexual differentiation. The prospective candidacy of select sexual-stage proteins as targets for transmission blocking strategies will be discussed.

Sex ratio theory posits that the adaptive proportion of male to female gametocytes of a malaria parasite within the vertebrate host depends on the degree of inbreeding within the vector. Gametocyte sex ratio could be phenotypically flexible, being altered based on the infection's clonal diversity, and thus likely inbreeding. This idea was tested by manipulating the clonal diversity of infections of Plasmodium mexicanum in its lizard host, Sceloporus occidentalis. Naive lizards were inoculated with infected blood from a single donor or 3 pooled donors. Donors varied in their gametocyte sex ratios (17–46% male), and sex ratio theory allowed estimation of the clonal diversity within donor and recipient infections. Phenotypic plasticity would produce a correlation between donor and recipient infections for infections initiated from a single donor, and a less female-biased gametocyte sex ratio in recipients that received a mixed blood inoculum (with predicted higher clonal diversity) than recipients receiving blood from a single donor. Neither pattern was observed. Gametocyte sex ratio of most infections ranged from 35 to 42% male, expected if clonal diversity was high for all infections. Alternative explanations are suggested for the observed variation of gametocyte sex ratio among P. mexicanum infections.

The sex ratios of malaria and related Apicomplexan parasites play a major role in transmission success. Here, we address 2 fundamental issues in the sex ratios of the rodent malaria parasite, Plasmodium chabaudi. First we test the accuracy of empirical methods for estimating sex ratios in malaria parasites, and show that sex ratios made with standard thin smears may overestimate the proportion of female gametocytes. Secondly, we test whether the mortality rate differs between male and female gametocytes, as assumed by sex ratio theory. Conventional application of sex ratio theory to malaria parasites assumes that the primary sex ratio can be accurately determined from mature gametocytes circulating in the peripheral circulation. We stopped gametocyte production with chloroquine in order to study a cohort of gametocytes in vitro. The mortality rate was significantly higher for female gametocytes, with an average half-life of 8 h for female gametocytes and 16 h for male gametocytes.

An epidemiological survey was conducted during a 4-month period of intense malaria transmission in Dielmo, a holoendemic Senegalese village. Two thick blood smears per inhabitant were collected weekly. The sex ratio of Plasmodium falciparum gametocytes (gamete precursors) was studied in 50 gametocyte carriers. All age classes were represented (mean 19·7 years; range: 2 months–75 years); 42 (84%) of them did not receive antimalarial treatment. Overall 668 thick smears were examined until 100 gametocytes had been counted or for 40 min. A total of 11 204 gametocytes were observed with a mean sex ratio of 0·346 (95% CI 0·317–0·374), i.e. 2·89 females per 1 male. Among the 284 thick smears in which at least 10 gametocytes were observed, the mean percentage of male gametocytes was 27·8%, with a range of 0–82%. Great variability was observed between gametocyte carriers and also between thick smears from the same gametocyte carrier. A multivariate analysis was performed which highlighted the fact that only 2 variables had a significant effect on the sex ratio. Anaemia was associated with an increased percentage of males (Prevalence Rate Ratio [PPR] of male gametocytes was multiplied by 1·65 if haematocrit rate <32%) and a wave of gametocytes was associated with an increased percentage of female gametocytes (PRR was multiplied by 0·48 during the peak of gametocytaemia and for the 2 weeks following this peak). The variables without significant effect on sex ratio were: age, sex, clinical status and sickle cell trait status of the gametocyte carrier, density of asexual parasites, quinine treatment, and gametocyte density (when taking account of its waves). These results are discussed in regard of possible differential production, mortality or sequestration of one gametocyte sex and selective advantages for the transmission of parasites.

To investigate the quantitative distribution of Plasmodium falciparum gametocytes into the vector bloodmeal, Anopheles arabiensis mosquitoes were fed on 3 volunteers naturally infected with gametocytes. The content of each mosquito midgut was smeared on a microscope slide and Giemsa stained. The distribution of gametocytes ingested by mosquitoes followed a negative binomial distribution, with apparently constant overdispersion (parameter k±S.E. = 3·105±0·392) for the 3 series. This aggregation of gametocytes in some midguts probably facilitates the conjunction of gametes and fertilization. This suggests that P. falciparum gametocytes in the peripheral blood flow of infected man do not follow an independent, homogeneous pattern but show a significant aggregation.