Revealing the molecular determinants of gender in malaria parasites

Malaria parasites have a complex life cycle with asexual multiplication in a vertebrate host and obligate sexual reproduction in the mosquito; however, commitment to sexual development begins in the vertebrate with differentiation of female and male gametocytes. In this issue of Cell, Khan et al. (2005) used elegant approaches to purify male and female gametocytes and elucidated their respective proteome, providing the basis for understanding sexual development in this pathogen.     

A developmental defect in Plasmodium falciparum male gametogenesis

Asexually replicating populations of Plasmodium parasites, including those from cloned lines, generate both male and female gametes to complete the malaria life cycle through the mosquito. The generation of these sexual forms begins with the induction of gametocytes from haploid asexual stage parasites in the blood of the vertebrate host. The molecular processes that govern the differentiation and development of the sexual forms are largely unknown. Here we describe a defect that affects the development of competent male gametocytes from a mutant clone of P. falciparum (Dd2). Comparison of the Dd2 clone to the predecessor clone from which it was derived (W2'82) shows that the defect is a mutation that arose during the long-term cultivation of asexual stages in vitro. Light and electron microscopic images, and indirect immunofluorescence assays with male-specific anti-alpha- tubulin II antibodies, indicate a global disruption of male development at the gametocyte level with at least a 70-90% reduction in the proportion of mature male gametocytes by the Dd2 clone relative to W2'82. A high prevalence of abnormal gametocyte forms, frequently containing multiple and unusually large vacuoles, is associated with the defect. The reduced production of mature male gametocytes may reflect a problem in processes that commit a gametocyte to male development or a progressive attrition of viable male gametocytes during maturation. The defect is genetically linked to an almost complete absence of male gamete production and of infectivity to mosquitoes. This is the first sex-specific developmental mutation identified and characterized in Plasmodium.     

Sexual differentiation and development in the malaria parasite

The protozoan parasites belonging to the genus Plasmodium have a complex life cycle in which the asexual multiplication of parasites in the vertebrate host alternates with an obligate sexual reproduction in the mosquito. Gametocytes (male and female) produced in the vertebrate host are responsible for transmitting parasites to mosquitoes. Although our understanding of the biology and genetics of sexual differentiation in Plasmodium is expanding, the most basic questions concerning molecular mechanisms of sexual differentiation and sex determination still remain unanswered. Recently, insight into the control of this complex process in P. falciparum and P. berghei has come from studying parasite mutants with aberrant capacities for gametocyte production. Here, Cheryl-Ann Lobo and Nirbhay Kumar review these analyses in P. falciparum.     

Parthenogenetic female populations in the brown alga Scytosiphon lomentaria (Scytosiphonaceae,Ectocarpales): decay of a sexual trait and acquisition of asexual traits

In isogamous brown algae, the sexuality of populations needs to be tested by laboratory crossing experiments, as the sexes of gametophytes are morphologically indistinguishable. In some cases, gamete fusion is not observed and the precise reproductive mode of the populations is unknown. In the isogamous brown alga Scytosiphon lomentaria in Japan, both asexual (gamete fusion is unobservable) and sexual populations (gamete fusion is observable) have been reported. In order to elucidate the reproductive mode of asexual populations in this species, we used PCR‐based sex markers to investigate the sex ratio of three asexual and two sexual field populations. The markers indicated that the asexual populations consisted only of female individuals, whereas sexual populations are composed of both males and females. In culture, female gametes of most strains from asexual populations were able to fuse with male gametes; however, they had little to no detectable sexual pheromones, significantly larger cell sizes, and more rapid parthenogenetic development compared to female/male gametes from sexual populations. Investigations of sporophytic stages in the field indicated that alternation of gametophytic and parthenosporophytic stages occur in an asexual population. These results indicate that the S. lomentaria asexual populations are female populations that lack sexual reproduction and reproduce parthenogenetically. It is likely that females in the asexual populations have reduced a sexual trait (pheromone production) and have acquired asexual traits (larger gamete sizes and rapid parthenogenetic development).     

Egress of Plasmodium berghei gametes from their host erythrocyte is mediated by the MDV-1/PEG3 protein

Malaria parasites invade erythrocytes of their host both for asexual multiplication and for differentiation to male and female gametocytes – the precursor cells of Plasmodium gametes. For further development the parasite is dependent on efficient release of the asexual daughter cells and of the gametes from the host erythrocyte. How malarial parasites exit their host cells remains largely unknown. We here report the characterization of a Plasmodium berghei protein that is involved in egress of both male and female gametes from the host erythrocyte. Protein MDV-1/PEG3, like its Plasmodium falciparum orthologue , is present in gametocytes of both sexes, but more abundant in the female, where it is associated with dense granular organelles, the osmiophilic bodies. Δ mdv-1/peg3 parasites in which MDV-1/PEG3 production was abolished by gene disruption had a strongly reduced capacity to form zygotes resulting from a reduced capability of both the male and female gametes to disrupt the surrounding parasitophorous vacuole and to egress from the host erythrocyte. These data demonstrate that emergence from the host cell of male and female gametes relies on a common, MDV-1/PEG3-dependent mechanism that is distinct from mechanisms used by asexual parasites.     

Isolation of early genes expressed in reproductive organs of the dioecious white campion (Silene latifolia) by subtraction cloning using an asexual mutant

The dioecious white campion (Silene latifolia) has been chosen as a working model for sexual development. In this species, sexual dimorphism is achieved through two distinct developmental blocks: inhibition of carpel development in male flowers, and early arrest of anther differentiation in female flowers. The combined advantages of the dioecious system and the availability of a sexual mutant lacking both male and female reproductive organs have been exploited in a molecular subtraction approach using male and asexual flower buds. This resulted in the cloning of 22 cDNA clones expressed in stamens at distinct stages of development. Fourteen of these clones corresponded to genes whose expression was detected in pre-meiotic stamens, a stage of development for which very little information is presently available. Furthermore, the absence of similarities with database sequences for ten clones suggests that they represent novel genes. Functional analysis of each clone will enable their positioning within the reproductive organ developmental pathway(s). In parallel, these clones are being exploited as developmental markers of early differentiation within the flower.     

An atypical mitogen-activated protein kinase controls cytokinesis and flagellar motility during male gamete formation in a malaria parasite

The transmission of malaria parasites to the mosquito depends critically on the rapid initiation of sexual reproduction in response to triggers from the mosquito midgut environment. We here identify an essential function for an atypical mitogen-activated protein kinase of the rodent malaria parasite Plasmodium berghei, Pbmap-2, in male sexual differentiation and parasite transmission to the mosquito. A deletion mutant no longer expressing the Pbmap-2 protein develops as wild type throughout the asexual erythrocytic phase of the life cycle. Gametocytes, the sexual transmission stages, form normally and respond in vitro to the appropriate environmental cues by rounding up and emerging from their host cells. However, microgametocytes fail to release flagellated microgametes. Female development is not affected, as judged by the ability of macrogametes to become cross-fertilized by microgametes from a donor strain. Cellular differentiation of Pbmap-2 KO microgametocytes is blocked at a late stage of male gamete formation, after replication and mitoses have been completed and axonemes have been assembled. These data demonstrate a function for Pbmap-2 in initiating cytokinesis and axoneme motility, possibly downstream of a cell cycle checkpoint for the completion of replication and/or mitosis, which are extraordinarily rapid in the male gametocyte.     

COMPARATIVE STUDIES ON THE CELL WALLS OF SEXUAL AND ASEXUAL BANGIA ATROPURPUREA (RHODOPHYTA). I. HISTOCHEMISTRY OF POLYSACCHARIDES1

A comparative histochemical study of the nature and distribution of acidic and neutral cell wall polysaccharides was conducted on marine sexual and asexual filaments of the red alga Bangia atropurpurea (Roth) C. Ag. Outer and inner walls of this species were clearly partitioned according to staining and transmission electron microscopic characteristics. Neutral polysaccharides were detected in the outer coating (cuticle) but were absent from outer and inner walls of all filaments. Acidic polysaccharides were noted in the outer wall material but not in the inner wall layers of any filaments at any developmental stages. The major staining component of vegetative regions of sexual material and all regions of asexual filaments was a highly sulfated polymer. During sexual reproduction only there was a generalized change in the nature of the acidic component, characterized by a decrease in intensity of staining for sulfates in both male and female filaments and the appearance, in female filaments only, of polysaccharides which presumably were carboxylated. Spermatia attached to both male and female filaments in regions where sexual differentiation was initiated and where changes in the outer wall components commenced.     

The maintenance of sex in parasites

The maintenance of sex is an unresolved paradox in evolutionary biology, given the inherent twofold fitness advantage for asexuals. Parasitic helminths offer a unique opportunity to address this enigma. Parasites that can create novel antigenic strains are able to escape pre-existing host immunity. Viruses produce diversity through mutation with rapid clonal proliferation. The long generation times of helminth parasites prevent them from adopting this strategy. Instead, we argue that sexual reproduction enables parasitic helminths to rapidly generate strain diversity. We use both a stochastic, individual-based model and a simple analytical model to assess the selective value of sexual versus asexual reproduction in helminth parasites. We demonstrate that sexual reproduction can more easily produce and maintain strain diversity than asexual reproduction for long-lived parasites. We also show that sexual parasite populations are resistant to invasion by rare asexual mutants. These results are robust to high levels of cross-immunity between strains. We suggest that the enhancement of strain diversity, despite stochastic extinction of strains, may be critical to the evolutionary success of sex in long-lived parasites.     

Host cell preference and variable transmission strategies in malaria parasites

Malaria and other haemosporin parasites must undergo a round of sexual reproduction in their insect vector in order to produce stages that can be transmitted to vertebrate hosts. Consequently, it is crucial that parasites produce the sex ratio (proportion of male sexual stages) that will maximize the number of fertilization and thus, transmission to new vertebrate hosts. There is some evidence to show that, consistent with evolutionary theory, the sex ratios of malaria parasites are negatively correlated to their inbreeding rate. However, recent theory has shown that when fertilization success is compromised, parasites should respond by increasing their investment in sexual stages or by producing a less female biased ration than predicted by their inbreeding rate alone. Here, we show that two species of rodent malaria, Plasmodium chabaudi and Plasmodium vinckei petteri, adopt different strategies in response to host anaemia, a factor though to compromise transmission success: P. chabaudi increases investment in sexual stages, whereas P. vinckei produces a less female biased sex ratio. We suggest that these different transmission strategies may be due to marked differences in host cell preference.     

The Plasmodium falciparum, Nima-related kinase Pfnek-4: a marker for asexual parasites committed to sexual differentiation

ABSTRACT: BACKGROUND: Malaria parasites undergo, in the vertebrate host, a developmental switch from asexual replication to sexual differentiation leading to the formation of gametocytes, the only form able to survive in the mosquito vector. Regulation of the onset of the sexual phase remains largely unknown and represents an important gap in the understanding of the parasite's complex biology. METHODS: The expression and function of the Nima-related kinase Pfnek-4 during the early sexual development of the human malaria parasite Plasmodium falciparum were investigated, using three types of transgenic Plasmodium falciparum 3D7 lines: (i) episomally expressing a Pfnek-4-GFP fusion protein under the control of its cognate pfnek-4 promoter; (ii) episomally expressing negative or positive selectable markers, yeast cytosine deaminase-uridyl phosphoribosyl transferase, or human dihydrofolate reductase, under the control of the pfnek-4 promoter; and (iii) lacking a functional pfnek-4 gene. Parasite transfectants were analysed by fluorescence microscopy and flow cytometry. In vitro growth rate and gametocyte formation were determined by Giemsa-stained blood smears. RESULTS: The Pfnek-4-GFP protein was found to be expressed in stage II to V gametocytes and, unexpectedly, in a subset of asexual-stage parasites undergoing schizogony. Culture conditions stimulating gametocyte formation resulted in significant increase of this schizont subpopulation. Moreover, sorted asexual parasites expressing the Pfnek-4-GFP protein displayed elevated gametocyte formation when returned to in vitro culture in presence of fresh red blood cells, when compared to GFP- parasites from the same initial population. Negative selection of asexual parasites expressing pfnek-4 showed a marginal reduction in growth rate, whereas positive selection caused a marked reduction in parasitaemia, but was not sufficient to completely abolish proliferation. Pfnek-4- clones are not affected in their asexual growth and produced normal numbers of stage V gametocytes. CONCLUSIONS: The results indicate that Pfnek-4 is not strictly gametocyte-specific, and is expressed in a small subset of asexual parasites displaying high rate conversion to sexual development. Pfnek-4 is not required for erythrocytic schizogony and gametocytogenesis. This is the first study to report the use of a molecular marker for the sorting of sexually-committed schizont stage P. falciparum parasites, which opens the way to molecular characterization of this pre-differentiated subpopulation.     

Functional characterisation of sexual stage specific proteins in Plasmodium falciparum

The various stages of the malaria parasites in the vertebrate host and in the mosquito vector offer numerous candidates for vaccine and drug development. However, the biological complexity of the parasites and the interaction with the immune system of the host continue to frustrate all such efforts thus far. While most of the targets for drug and vaccine design have focused on the asexual stages, the sexual stages of the parasite are critical for transmission and maintenance of parasites among susceptible vertebrate hosts. Sexual stage parasites undergo a series of morphological and biochemical changes during their development, accompanied by a co-ordinated cascade of a distinct expression pattern of sexual stage specific proteins. Mechanisms underlying the developmental switch from asexual parasite to sexual parasite still remain elusive. Methods that can break the malaria transmission cycle thus occupy a central place in the overall malaria control strategies. This paper provides a review of genes expressed in sexually differentiated Plasmodium. In the past few years, a molecular approach based on targeted gene disruption has revealed fascinating biological roles for many of the sexual stage gene products. In addition, we will briefly discuss other functional genomic approaches employed to study not only sexual but also other aspects of host-parasite biology.     

THE GENETIC BASIS OF MALE POTENCY IN VOLVOX CARTERI F. NAGARIENSIS (CHLOROPHYCEAE)1,2

Sexual males of Volvox carteri Stein f. nagariensis Iyengar secrete a hormone which induces sexual rather than asexual development in the next generation. Male recombinants from the original strains isolated from Japan showed an increase in potency of up to 100 times that of the original male clone, with later descendants showing potencies ranging from 100 times to 1/1000 the potency of this male. Potency is defined as the extent to which the hormone produced in 1 ml of medium by one parental spheroid containing 16 male spheroids can be diluted and retain its capacity to induce sexual differentiation. This is recorded as the percent of female response in the tube of limiting dilution, asexual females of the normal female strain being used as assay inoculum in a 1 in 10 dilution series of samples of male fluid containing the sexual hormone. Investigation of the genetic basis for potency in this species included selection of high and low potency male clones and crossing with sib females or backcrossing with original female. Results of the various crosses are interpreted as multigenic system not linked with sex, with more than one genotypic class per dilution level and with more than one genotypic class per apparent phenotypic class. Possible genic interactions are described in their relevance to the biosynthesis of a glycoprotein molecule, the sexual hormone having been characterized as a glycoprotein by Starr and Jaenicke (18).     

栝楼不同性别花芽分化形态解剖特征观察

采用体视显微镜、石蜡切片和树脂切片技术对栝楼(Trichosanthes kirilowii Maxim.)不同性别花芽分化发育时期的外部形态和内部解剖结构进行了观察。结果显示,栝楼花为雌雄异株,仅有雌花、雄花两种性别分化,且雄花的发育速度明显快于雌花的发育速度。栝楼雌雄花芽长0.2 mm左右已完成性别分化;栝楼雄花为单性花,分化过程可分为6个时期,整个发育过程仅见雄蕊原基的分化及生长。栝楼雌花为"两性花",分化过程可分为7个时期,存在雌蕊和雄蕊共同发育阶段,后期雄蕊发育败退。本研究明确了不同性别栝楼花芽发育发生的各个阶段、形态变化特点、外部形态变化特征以及雌雄花芽的分化差异,建立了雌雄花芽内部结构分化与外部形态之间相关性,为栝楼早期幼苗鉴定及性别分化研究提供了一定的参考。     

Density,parasitism, and sexual reproduction are strongly correlated in lake Daphnia populations

Many organisms can reproduce both asexually and sexually. For cyclical parthenogens, periods of asexual reproduction are punctuated by bouts of sexual reproduction, and the shift from asexual to sexual reproduction has large impacts on fitness and population dynamics. We studied populations of Daphnia dentifera to determine the amount of investment in sexual reproduction as well as the factors associated with variation in investment in sex. To do so, we tracked host density, infections by nine different parasites, and sexual reproduction in 15 lake populations of D. dentifera for 3 years. Sexual reproduction was seasonal, with male and ephippial female production beginning as early as late September and generally increasing through November. However, there was substantial variation in the prevalence of sexual individuals across populations, with some populations remaining entirely asexual throughout the study period and others shifting almost entirely to sexual females and males. We found strong relationships between density, prevalence of infection, parasite species richness, and sexual reproduction in these populations. However, strong collinearity between density, parasitism, and sexual reproduction means that further work will be required to disentangle the causal mechanisms underlying these relationships.     

Interference of asexual and sexual reproduction in the green hydra

The green hydra, Hydra viridissima, has three sexes: hermaphrodite, male, and female. I investigated the reproductive strategies of the green hydra and the relationship between asexual budding and sexual reproduction. The proportion of mature individuals in the asexually reproducing population increased with increasing temperature. Sexual reproduction did not interrupt asexual budding in hermaphrodites or males; sexual reproduction did interrupt asexual budding in females. Sexual reproduction also resulted in exponential population growth during the reproductive season. The number of asexual buds on each parental individual was positively correlated with the parental individual size in asexual individuals and in males. The same positive correlation was found between the number of testicles and the size of males. These correlations reflect a common tendency in asexual and sexual reproduction: larger parental individuals have a greater number of propagules or gametes. No correlation was found between size and buds or size and gonads in hermaphrodites; hermaphrodites had at most one asexual bud and were significantly larger than males, females, and asexual individuals. The larger size of hermaphrodites supports the hypothesis that producing both female and male gonads is more energetically costly than producing only one type of gamete (gonochorism).     

Plasmodium falciparum: mRNA co-expression and protein co-localisation of two gene products upregulated in early gametocytes

Genes encoding Plasmodium falciparum proteins Pfs16 and Pfpeg3/mdv1, specifically appearing in the parasitophorous vacuole of the early gametocytes, are upregulated at the onset of sexual differentiation. Analysis of asexual development in gametocyte producing and non-producing clones of P. falciparum indicated that these genes are also transcribed at a low level in asexual parasites, although their protein products are not detectable in these stages by immunofluorescence. Immunoelectron microscopic analysis of stage II gametocytes indicated that Pfs16 and Pfpeg3/mdv1 proteins co-localise in the parasitophorous vacuole membrane and in all derived membranous structures (such as the multi-laminate membrane whorls of the circular clefts in the infected erythrocyte cytoplasm and the membranes of the gametocyte food vacuoles). In this analysis both proteins were also observed for the first time in the membrane and in the lumen of distinct cleft-like structures in the erythrocyte cytoplasm.