Sexual differentiation in malaria parasites.

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SEXU'AL DIFFERENTIATION IN Annu. Rev. Microbiol. 1990.44:429-449. Downloaded from www.annualreviews.org by Lomonosov Moscow State University on 02/20/14. For personal use only.

MAL1\RIA PARASITES Pietro Alana and Richard Carter Department of Genetics, University of Edinburgh, Edinburgh, Scotland KEY WORDS:

gametocytes, gametes, plasmodium, ribosomes, tubulin

CONTENTS INTRODUCTION,.................................................................................... DIFFERENTIATION OF SEXUAL AND ASEXUAL MALARIA PARASITES IN THE VERTEBRATE HOST.................................................................. The Origin of Gametocytes . . , . . . . . . . . ... . . . . . . . ..... . . . . . . . .... . . . . . ....... . . ....... . . . ...... .. Commitment to Gametocyte Production.... .. . .... . . . .. . . ........ . . ...... . . . .. .... . . ...... .. . Modulation of Gametocyte Production ... .. .. . . . ..... . . ... . . . . ...... . .... .. . . . . . . . . . . ... ... . . Genetic Basis of Gametocyte Production .. . ............ ............................ . ........ SEX DETERMINATION IN MALARIA PARASITES .. . . . . . . ... . . . . . ... . . . . . . . . ... . . . ......

429 431 431 431 433 435 437

TRANSFORMATION OF SEXUAL STAGES OF MALARIA PARASITES IN THE MOSQUITO VECTOR ..........................................................

438

SEXUAL STAGE MALARIA PARASITES AS SPECIALIZED CELLS ................. Cytoskeleton and Microtubular Organization ..... . .................... ..................... Ribosomes .. ... ... ....... .. .. . . ..... . .. ...... . . . . ... . . . . ... . . ..... .. . . . . . . ..... . . ... ....... .. . .. .... Nucleus, Chromosomes, and DNA Content.. . . . . . . . ..... .. . ........ . . .. . ...... . .... .. . . .. ... Expression of Sexual Stage-Specijic Proteins . . .. . . .. .. . . . . ........ . .. . . ..... ...... ... . .. . ..

440 441 441

CONCLUSIONS.......................................................................................

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443 445

INTRODUCTION

The Plasmodia, or malaria parasites, are parasitic protozoa that infect a wide range of vertebrate hosts (35) including reptiles, birds, rodents, and primates; in humans, they are the agents of malaria, one of the most prevalent human infectious diseases. The Plasmodia belong to a group of parasites, the order Haemosporina, that infect the blood of various vetebrates. The Haemosporina share most features of the malaria parasites' life cycle (Figure 1). All includ429

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Annu. Rev. Microbiol. 1990.44:429-449. Downloaded from www.annualreviews.org by Lomonosov Moscow State University on 02/20/14. For personal use only.

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ALANO & CARTER

IN MOSQUITO GUT

Figure 1

Life cycle of a malaria parasite, PlasmodiumJalciparum. Sporozoites in the salivary

glands of a female mosquito are inoculated into the bloodstream during a blood meal. The sporozoites invade the liver parenchyma cells where they develop as intracellular hepatic schizonts. After about six days, the schizont ruptures, releasing several thousand merozoites that invade circulating red blood cells. In the blood, the parasites can develop either as asexual parasites (each fonning a blood schizont producing up to 32 merozoites) or as sexual parasites. These male and female gametocytes are the only fonns able to survive in the mosquito midgut following a blood meal. Within the mosquito midgut, gametocytes transfonn into gametes and fertilize; the newly fonned zygote transfonns into an ookinete, a motile stage that crosses the midgut wall and develops into an oocyst on the outer side of the gut. The oocyst grows over 10 to

14 days, producing sporozoites that migrate to the salivary glands, ready to enter the vertebrate blood steam during a subsequent blood meal. (From Ref. 39, with pennission.)


MALARIA PARASITE DIFFERENTIATION

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ing the Leucocytozoidae (e.g. Leucocytozoon simondi, a parasite of the white blood cells of ducks and geese) and the Haemoproteidae (e.g. Haemoproteus columbae, a parasite of red blood cells of pigeons) form gametocytes in the circulating blood cells. Only true malaria parasites (family Plasmodiidae, genus Plasmodium), however, undergo asexual multiplication (schizogony) in the blood. The choice to differentiate as either sexual or asexual forms in the blood is, therefore, unique to the genus Plasmodium. Comprehensive reviews on the sexual stages of malaria parasites (15, 81) and on sex determination and differentiation in malaria parasites (22) have been published. The present review concentrates on the processes of differen tiation that characterize the origin, growth, and development of sexual-stage malaria parasites in the vertebrate host and in the mosquito vector. DIFFERENTIATION OF SEXUAL AND ASEXUAL MALARIA PARASITES IN THE VETEBRATE HOST

The Origin of Gametocytes As do other members of the Haemosporina, gametocytes of Plasmodium can arise from merozoites from asexual stages that precede parasitization of the blood. In the human malaria parasite Plasmodium vivax (45) and the rodent malaria parasite Plasmodium yoelii (57), gametocytes are present following sporozoite-induced infections in less than the time possible for them to have derived from a round of asexual development in the blood. Such gametocytes could only come from merozoites from liver-stage parasites. A study of the bird malaria parasite, Plasmodium gallinaceum, showed that gametocytes form from pre-erythrocytic schizonts in relapse infections after primary blood infection is terminated with quinine (1). Neverthdess, during natural infections, most gametocytes arise from merozoites of blood-stage origin. This source of gametocytes is formally proved by the presence of gametocytes in blood-induced infections and in in vitro culture of blood-stage malaria parasites (e.g. 19, 51, 69). Commitment to Gametocyte Production In early speculations on the differentiation of sexual and asexual forms of P. vivax (9,25) and another human malaria parasite, Plasmodium malariae (10), two different kinds of blood schizont were postulated, one producing only gametocytes and the other only asexual parasites. Aikawa et al (4) suggested an alternative idea: that the merozoites are not precomitted in the schizont but can differentiate into either sexual or asexual forms on reinvasion. These speculations imply two alternative general possibilities by which malaria parasites could become committed to either sexual or asexual development, as summarized in Figure 2.

432

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Models for gametocytogenesis in Plasmodium. Modell; the merozoite is not com

mitted at the time of invasion of a red blood celL During early growth (as a ring form), the parasite is susceptible to factors that will commit it to either sexual or asexual development.

Model II; during growth of an asexual parasite, environmental factors influence it so that at maturity the schizont produces merozoites that are committed to form asexual parasites or committed to form gametocytes upon invasion of a red blood cell. (From Ref. 1 9, with permission.)

Inselburg (43) reported the first controlled experiment on this subject. Plasmodiumfalciparum schizonts were allowed to rupture on a monolayer of uninfected red blood cells bound by concanavalin A to the bottom of a plastic Petri dish. Merozoites released from individual schizonts invaded adjacent red blood cells in the monolayer, forming a plaque of daughter parasites. Ninety six hours after invasion, the infected monolayers were stained and the dis tribution of sexual and asexual parasites was determined on the basis of their ' morphology. Inselburg (43) concluded that some schizonts' exhibited a marked tendency to produce gametocytes. However, the commitment to gametocytogenesis by such schizonts was not absolute because most of the gametocyte-containing plaques also contained asexual parasites. This conclu sion represents a modification of Model II proposed in Figure 2. The question has been re-examined with a similar assay using stage specific monoclonal antibodies (Mabs) to distinguish gametocytes and asex ual-stage parasites (13). Infected monolayers were reacted at 30 to 40 hours


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post invasion with a Mab recognizing a schizont stage-specific protein, MSA-2 (33), and another directed against a sexual stage-specific protein, Pfg 27125 (16). The stage specificity of parasites in each plaque was determined in a double immunofluorescence assay (lFA) u sing a different fluorochrome for


each Mab. In this study, parasites derived from individual schizonts were found to be either all gametocytes or all asexual parasites. This result strongly im pl ies that merozoites from an individual schizont of P. Jalciparum are already committed to one or the other developmental pathway before entering a red blood cell. The commitment by the progeny of individual schizonts is virtually absolute, as proposed in Model II, Figure 2. This degree of commit

ment contrasts with the result of Inselburg (43). Whether the discrepancy results from differences in experimental conditions, the parasite lines used, or the methods of identifying the parasites in each study is not clear. Commitment to sexual differentiation has also been studied in the rodent malaria parasite Plasmodium berghei. The experimental system involved

synchroniza.tion of the parasites (68, 69) so that the asexual forms were at the same stage of development at any point in time. The synchronized parasites were introduced into rats and allowed to grow and reinvade red blood cells in vivo. At different times during the infection, parasites were taken from the rat, placed in in vitro culture and monitored for gametocyte formation. Parasites introduced into culture shortly after merozoites had invaded red blood cells in the rat failed to form gametocytes but developed into asexual

stages. Parasites introduced into culture midway through their growth formed gametocytes in vitro in similar proportions to those remaining in the host. These results seemed to indicate that conditions in the rat influenced certain of the parasites midway through their growth to become gametocytes, whereas such conditions are absent from in vitro cultures. These conclusions imply

that the young parasite, shortly after entering a red blood cell, is neither sexual nor asexual and can be influenced to enter either path of development. In contrast to that proposed for P. Jalciparum, this interpretation corresponds to Model I, Figure 2 (13). Wh et her this behavior represents a true difference between the parasite species or their conditions of growth or results from artifacts of experimentation is again not clear.

Modulation of Gametocyte Production The proportion of parasites that de velop into gametocytes varies greatly during the course of natural malarial infections (85, 87). Sev eral attempts have been made to study this process in vitro and in vivo. Using stage-specific Mabs, as described above, with a clone of P. Jalciparum grown in culture, researchers have confirmed that changes in conditions of growth of the asexual parasites are associated with large-scale changes in the proportion of merozoites that form gametocytes (13). At high parasite densities, a high


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proportion of merozoites formed gametocytes; dilution with fresh red blood cells caused a dramatic decrease (up to lOO-fold) in the proportion of parasites that transformed into sexual forms. Changes from high to low gametocyte conversion rates following addition of fresh red blood cells occurred within less than the time required for one cycle of asexual development (48 hours). Thus, the growing asexual parasite in the period preceding schizogony is directly responsive to the conditions that modulate gametocyte production. Therefore, commitment to sexual or asexual development in the daughter merozoites is made at this stage, as proposed in Model II, Figure 2. A number of studies have attempted to identity factors that may control the differentiation of sexual- and asexual-stage malaria parasites. Among these, controversy has surrounded the role of cyclic adenosine monophosphate (cAMP), since an increase in gametocyte conversion rates in cultures of P. Jalciparurn was reported (55). Similar but less pronounced effects have been reported (81), but other investigators have been unable to reproduce such results (12, 44, 93). Several reports showed that cAMP, at certain con centrations, can inhibit growth of young asexual parasites (44, 55, 81). Other compounds affecting metabolism of cAMP have been reported to stimulate gametocytogenesis in cultures of P. Jalciparurn. These two inhibitors of the enzyme cAMP phosphodiesterase, namely caffeine (12) and 8-bromo cAMP, were used at concentrations below those that inhibit asexual growth (93). Some phorbol esters, phorbol 12-myristate 13-acetate and phorbol 12, 13dibutyrate, were reported to increase gametocyte numbers in culture (93), as were corticosteroids (63, 89). But in another study, hydrocortisone did not have any effect (93). Pronounced effects on the production of gametocytes in vivo have been reported following treatment with certain classes of antimalarial drugs. Sever al reports record increased numbers of circulating gametocytes in P. Jalcipar urn patients following treatment with pyrimethamine and proguanil, both inhibitors of dihydrofolate reductase, and with sulfadiazine, a competitive inhibitor of para-amino benzoic acid metabolism (34, 60, 76, 79); all these drugs interfere with folate metabolism and ultimately nucleic acid synthesis. Patients treated with quinine, with a 4-aminoquinoline (mepacrine), or with an 8-aminoquinoline (pamaquine) did not have increased numbers of gameto cytes. Many early workers concluded that acquisition of immunity led to suppres sion of gametocyte production in human malaria (see reviews in 6 and 35). Infections in individuals with high levels of immunity generally produce fewer gametocytes than do those in nonimmune individuals (e.g. 21, 67). This difference would be expected from the fact that average densities of asexual parasites, from which gametocytes are derived, are also much lower in immune than in nonimmune persons. Some evidence suggests that immu-



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nity may, nevertheless, increase the rate of conversion of asexual parasites to gametocytes. In blood cultures of P. Jalciparum, conversion to gametocytes was stimulated in the presence of lymphocytes and serum from individuals with acquired immunity to P. falciparum (86). Similar results were obtained with P. falciparum grown in culture medium to which sera containing anti malarial antibodies was added (11) or in culture medium supplemented with supernatants from hybridoma cell cultures containing antimalarial antibodies (73). Because other factors present in such additives cannot be excluded from exerting these effects, the possible role of immunity or of antimalarial anti bodies in modulating gametocyte production remains unclear. A final (�xample of conditions that affect gametocyte production is associ ated with transfer of malaria parasites between hosts of different species as has been demonstrated with the rodent malaria parasite Plasmodium cha baudi. Thi s parasite grows to high parasitaemias in mice but few gametocytes are produc1ed; if, however, the parasites are introduced into splenectomised rats, a large proportion of the parasites become gametocytes (61). A study on a cloned hne of P. chabaudi showed that increased gametocyte numbers resulted from an increase in the gametocyte conversion rate in the rats and not from differential survival of sexual and asexual forms (24). Gametocytes and asexual parasites were present in both rat and mouse erythrocytes recovered from the rat blood stream, which appears to exclude the possibility that sexual or asexual development was determined by the type of host cell that a parasite entered and suggests that other environmental factors in the rat favored the induction of gametocytes. ,

Genetic Basis of Gametocyte Production As discussed above, cloned lines of malaria parasites can produce both asexual and sexual forms and indeed gametocytes of both sexes (5, 7, 14, 32, 68, 90, 98). Also well established is that malaria parasites are haploid throughout almost their entire life cycle from the sporozoite stage through the asexual fonns in the blood, leading to the formation of the gametocytes (99, 101). Therefore, differences between sexual and asexual malaria parasites, or indeed between gametocytes of different sexes, probably do not result from differences in genetic content arising from segregation of chromosomes. Consistent with this view is the failure to detect differences in the number or size of chromosome-sized DNA molecules extracted from asexual parasites and gametocytes of the same line of P. falciparum and analyzed on pulse field gradient electrophoresis (PFGE) (95). Experimental approaches to studying the genetic determinants underlying gametocyte production can take account of two well-established features of this phenomenon: (a) different lines of parasite within a Plasmodium species differ markedly in their intrinsic capacity to produce gametocytes, and (b)


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parasite lines propagated exclusively by asexual growth either in vitro or in vivo tend to progressivel y lose the capacity to produce gametocytes. Variabil ity in gametocyte production is found not only between parasites from differ ent patients [with P. jalciparum (12, 14, 36)] or animal isolates [with P. berghei (46, 68)] but also between clones derived from the same isolate of P. jalciparum (36). Moreover, parasite clones successively derived from a pri mary cloned line of P. jalciparum exhibit differences in their ability to produce gametocytes (D. Read & E. Rappocciolo, personal communication). The rate at which lines of malaria parasite lose their capacity to produce gametocytes varies but tends to last from several months to more than a year of continuous asexual multiplication (15). In a line of P. berghei, gametocyte

production decreased to about 30% of its starting capacity after 25 successive syringe passages in mice; capacity was almost entirely lost by the 150th passage (68), confirming previous findings (5). In P. jalciparum, gametocyte production may cease within a few months of continuous asexual multiplica tion (12, 36), be maintained for up to a year (19, 75, 92) before capacity

for gametocyte production is lost, or possibly be maintained indefinitely (93). The study of such parasite lines, which are in effect "mutants" defective in gametocyte production, could help identify the genes involved in th is produc tion. In principle, a parasite clone that forms normal gametocytes in reduced numbers is altered in its response to stimuli to enter commitment to gametocy

togenesis. A clone that totally fails to produce gametocytes could have entirely lost its ability to respond to such stimuli or it could be defective in the program governing the construction of a gametocyte. In most reported ex amples the loss of c ap acity was associated with the ,

production of fewer gametocytes that were, when tested, still functional and able to infect mosquitoes (5, 46, 68). In such parasites, the full genetic

information for completing gametocyte maturation must still have been present in at least some members of the parasite population. When such lines were transmitted through mosquitoes and into a vertebrate host, capacity for high levels of gametocyte production was restored [in Plasmodium cathemerium (42) and P. berghei (46, 68)]. This recovery could have resulted from selection of clones with high capacity during mosquito infection or from

reconstruction during meiosis in the mosquito, possibly as a consequence of hybridization and recombination between different clones of parasite genomes with intrinsically high capacity for gametocytogenesis. Lines of parasite have also been reported with apparent defects in their ability to construct a gametocyte. In P. berghei, lines with low gametocyte production often form gametocytes with degenerate morphology (46, 68). Lines of P. jalciparum have been described in which most gametocytes failed


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to develop beyond stage III (90; P. Graves, personal communication). Such mutants could form 'a basis for studying the genes responsible for the special ized structures of the gametocyte cell. For more information on the generation of genetic: changes in malaria parasites, see the article by McConkey et al (63a) in this volume.


Some studies have reported the possible molecular basis of gametocy

togenesis. In P. berghei, progressive loss of gametocyte production was suggested to accompany reduction in the amount of repetitive DNA in the parasite genome (5). Interpretation of these results was complicated by the presence of a c ontami nati ng population of a different species of rodent malaria parasite, P. yoelii. In sep arate experiments, however, the authors confirmed that some loss of repetitive DNA acc ompa nied serial asexual passage (20). Qualitative differences in chromosome-sized DNA molecules were observed with field inversion gel electrophoresis (FlGE) in clones of P. berghei derived after asexual passage and progressive loss of gametocyte production; cytofluorimetric analysis failed, however, to detect quantitative differences in DNA co nte nt between the lines (46).

SEX DETERMINATION IN MALARIA PARASITES As already discussed, cloned lines of malaria parasites produce both male and female gametocytes. In view of the haploid nature of blood sta ge parasites, -

the sex of a gametocyte probably does not result from genetic differences between male and female parasites. Indeed, just as the proportion of parasites

that become gametocytes fluctuates under different conditions, so, to a lesser extent, does the ratio of male and female gametocytes. In general, the number of female gametocytes predominates over the number of male (e.g. 78), but this predominance may vary at different times in the same or different infections with a parasite line. Little is known of the basis of commitment of gametocytes to be male or female. No reagents are available that distinguish male and female gameto cytes during early development. A study on L. simondi may, however, have data relevant to this question. Electron microscopy revealed two kinds of merozoites in schizonts committed to gametocyte production (30). Some schizonts contained only merozoites with de nse ly pac ked ribosomes, which were suggested to be precursors of female gametocytes; others contained mixtures of clearly distinguishable merozoites, some with densely packed ribosomes and others with few ribosomes. The latter were suggested to be precursors of male gametocytes. If this interpretation is correct, the sex of future garnetocytes is already determined in the merozoites within the schi zont. There is no sound evidence on this point in Plasmodium.

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TRANSFORMATION OF SEXUAL STAGES OF MALARIA PARASITES IN THE MOSQUITO VECTOR

The transfer of malaria parasites from the circulation of a vertebrate host to the midgut of a mosquito vector requires the parasites to respond rapidly to their new environment. Only the gametocytes are adapted to do so and their response is almost immediate; within seconds of entering a mosquito midgut the processes of gametogenesis are triggered and fertilization is largely com plete within 15 to 20 minutes (Figure 1). Gametogenesis in malaria parasites involves the simultaneous activation of several, apparently independent, sets of cellular events. These may be grouped as follows, (a) disruption of the host cell and release of the parasite (emergence), (b) the reorganization of cytoplasmic structures involved in the formation of the male gametes, and (c) the replication of the genome of the male gametocyte to provide eight gametes. The latter two sets of processes comprise the dynamic event classically referred to as exflagellation. This event and the ensuing processes of fertilization and ookinete formation have been described using both light and electron microscopy (for reviews, see 15, 81). The events of gametogenesis are tightly regulated under several levels of control. For most species of malaria parasite, gametocytes withdrawn from the circulation of the host spontaneously exflagellate on exposure to air. This phenomenon suggested that environmental changes associated with the para sites' transfer to ambient conditions mediated the initiation of gametogenesis. Both temperature and pH are critical in controlling the process. In addition, factors in the mosquito itself are probably essential for gametogenesis during the natural transmission of the parasites. The first carefully controlled experiments on gametogenesis were by Bishop & McConnachie (8), who demonstrated that the minimal components of blood plasma needed to support spontaneous exflagellation of the avian parasite, P. gallinaceum, are Na+ and HCOi ions. Later studies showed that emergence and exflagellation in a minimum solution of NaCl and NaHC03 (buffered with Tris and with glucose as an energy source, which is essential for gametocyte and gamete viability) were strongly pH dependent (71). Both processes were totally suppressed below pH 7.8 and were maximally stimu lated at pH 8.1. Above pH 8.5, exflagellation was again totally suppressed, although emergence was undiminished, demonstrating the independent con trol of these two processes. Both, however, entirely depended on the presence ,of bicarbonate ion; in its absence, neither event took place at any pH. Exposure of P. gallinaceum gametocytes to these permissive conditions for as little as ten seconds triggers the process into irreversible motion (15).



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A similar pH dependence of exflagellation has been demonstrated in the rodent malaria parasite P. berghei (F. Kawamoto & R. E. Sinden, personal communication), but bicarbonate ion was not found to be an essential com ponent. Tilli s system was used to study the significance of ion transport in the pH-mediated control of gametogenesis. The presence of amiloride, an in hibitor of Na+ -H+ exchange, prevented exflagellation under otherwise per missive conditions (culture medium at pH 8.0). Na+-H+ exchange may mediate a rise in internal pH of the gametocyte-infected red blood cell, thereby creating the intracellular conditions that trigger gametogenesis. Although amiloride prevented male gamete formation, the activated male gametocytes nevertheless continued to replicate DNA. Thus, only the cytoplasmic events of gamete formation appeared to be under the control of pH and ion exchange. Other pharmacological agents initiate gametogenesis. Inhibitors of phos phodiesterase such as 8-bromo 3' 5' cAMP and caffeine stimulated ex flagellation of gametocytes of P. gallinaceum in bicarbonate-free saline (62). Similar effects have not been found for P. berghei, but an inhibitor of cyclic guanosine monophosphate phosphodiesterase, 3-isobutyl-I-methylxanthine, enhanced exflagellation at pH 7.3. Antagonists of intracellular Ca+ + mobilization, such as TMB-8 and W-7 (a calmodulin inhibitor), inhibited exflagellation of P. berghei gametocytes under permissive conditions at pH 8.0 (56a). All the events of gametogenesis are under strict temperature control. Even under othe:rwise permissive conditions of pH, or in the presence of pharmaco logical activators of gametogenesis, gametocytes maintained at normal host body temperature do not undergo any of the processes of gametogenesis (15, 81). At a few degrees below body temperature, however, all three processes may be triiggered. The rates at which emergence and exflagellation proceed follow different temperature curves. Exflagellation, and probably DNA syn thesis, proceed at maximum rate, taking about ten minutes, at the highest permissivt� temperature (about 34°C for P. falciparum and about 38°C for P. gallinaceum, the chicken having a nonnal body temperature about 2°C higher than humans). Emergence, on the other hand, is relatively slow at the highest permissivt� temperature and proceeds at maximum speed, taking about ten minutes for P. gallinaceum, at typical tropical ambient temperatures of 24-26°C. Exflagellation is somewhat slower at these temperatures, taking about fifteen minutes, and thus allows emergence to be completed before the male gametes are fully formed and released. Both emergence and exflagella tion become progressively delayed at lower temperatures; exflagellation can not take place below lOoC, but emergence continues to completion at temper atures as low as 4°C, although it is delayed by up to one hour (15).


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Therefore, at least two types of control prevent gametogenesis in the host circulation-temperature control and a pH, or ionic, control. However, an other set of controlling factor(s) involves compounds present in, or released into, the blood meal of the mosquito vector. The pH of blood ingested by a mosquito rarely rises above pH 7.6 (8). Nevertheless, exflagellation takes place with great efficiency in a mosquito midgut, and gametocytes of P. gallinaceum exflagellate when fed to mos quitoes in a simple NaCl solution, without bicarbonate ion and buffered with Tris at pH 7.5 (70). The mosquito midgut contains a highly active factor, termed mosquito exflagellation factor (MEF), which, like the pharmacologic al agents discussed above, stimulates gametogenesis of P. gallinaceum via a bicarbonate- and pH-independent mechanism (70). MEF appears to be a small organic compound of less than Mr 500 (R. F. Beach & M. M. Nijhout, personal communication), which is also found in mosquito head tissue but not in ovaries or Malpighian tubules (70). The activity of such agent(s) is probably the primary mechanism by which gametogenesis is initiated in a mosquito blood meal under natural conditions. An extreme instance of dependence upon such a factor has been found in the bird malaria Plasmodium elongatum. Gametogenesis in this species does not take place spontaneously on exposure of gametocyte-infected blood to air but only in a vector mosquito such as Culex pipiens (66). Nonvector species such as Aedes aegypti or Anopheles quadrimaculatus do not allow exflagella tion to take place, apparently because of the presence of species-specific inhibitory factors. SEXUAL STAGE MALARIA PARASITES AS SPECIALIZED CELLS

The basic features of gametocytes are similar in all species of malaria parasite. Several, most notably those of P. Jalciparum, have atypical aspects. Because of the parasite's importance as a human pathogen and the availability of the sexual stages in blood-stage culture in vitro (94), the gametocytes of P. Jalciparum have been widely studied. Those grown in vitro appear in distinguishable from those found in vivo (51). The growth and differentiation of gametocytes of P. Jalciparum has been divided into five stages covering about eight days from merozoite invasion to mature gametocyte (40). Each stage is distinguished by successive changes in the organization of the cel\ as seen by light microscopy on blood smears stained with Geimsa's stain. Ultrastructural details of this process have been studied using electron micros copy (80) and reviewed elsewhere (15, 81). We discuss here features of cellular and molecular organization specific to the differentiating sexual stages of the parasites.


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Cytoskeleton and Microtubular Organization The most obvious characteristic of P. falciparum gametocytes is their elon gated shape within the host red blood cell. This becomes apparent two to three days after merozoite invasion of a red blood cell. At this time, the gameto cytes (stage II) appear as crescents, occupying half the volume of the red blood cell. The crescent shape of the stage II gametocytes is due to a network of subpelliicular microtubules subtending a bilamellar inner membrane; these features are the first ultrastructural elements diagnostic of young P. falcipar um gametocytes (80). The organization of the subpellicular membranes and adjacent microtubules gives the gametocytes the elongated shape and pointed ends that characterize them in the third to the sixth or seventh day of development (stages III and IV). Microtubule assembly is regulated during maturation because the mature gametocyte (stage V) no longer possesses the subpellicular microtubules. Microtubules and their precursor, tubulin, are also important in the nuclear and cytoplasmic events of gametogenesis during spindle formation and segregation of the nuclear genome and in axoneme formation during exflagellation. The molecular subunit of microtubules is a protein heterodimer consisting of a beta and an alpha tubulin. P. Jalciparum has one beta tubulin gene (28, 103) and two alpha tubulin genes whose products have been designated alpha tubulin I :md alpha tubulin II (41). The tubulin genes of P. Jalciparum are differentially expressed in asexual parasites and in gametocytes. A probe derived from the coding sequence of the beta tubulin gene hybridized with three different poly A + transcripts in Northern blot analysis of gametocytes but with only one major transcript in analysis of asexual parasites (c. F. Delves & P. Alano, unpublished results). The same analysis showed that the alpha I gene is transcribed in both asexual parasites and gametocytes, while the alpha][l gene is expressed predominantly in gametocytes. This interesting example shows that genes belonging to a single multigene family (tubulin genes) are controlled independently of each other during development. This type of control appears to be typical of P. Jalciparum, as illustrated in the examples discussed below. Other components of the structural filaments are differentially regulated in asexual parasites and gametocytes. Two actin genes have been described in P. Jalciparum; Pf-actin I is expressed in both asexual and sexual parasites, while the transcript of Pf-actin 2 is detectable only in gametes and zygotes (104).

Ribosomes High densities of ribosomes are present in developing gametocytes mainly in the form of polysomes (80). In the mature gametocytes, however, the ribo somal density is much lower in the male than in the female. The ribosomes of the male gametocyte are mainly organized in polysomes, and these gameto-


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cytes have no nucleolus. In female gametocytes, ribosomes are diffused as monomers throughout the cytoplasm; the presence of a nucleolus in the female nucleus is consistent with active ribosomal synthesis (2, 3, 77, 80, 88). Malaria parasites are unusual both in their organization and control of synthesis of ribosomal RNA (rRNA). In eukaryotes, a single rRNA transcrip tional unit codes for the 25S and the 5.8S rRNAs, which are assembled in the large ribosomal subunit, as well as the 18S rRNA, which is assembled in the small ribosomal subunit. In most organisms, the rRNA transcriptional unit is present in multiple copies, often organized in clustered tandem repeats. Moreover, these genes are amplified during oogenesis in many eukaryotic species (52). In P. Jalciparum, only four to six copies of the gene are found interspersed in the genome (64). Despite their low copy number, the rRNA of malaria parasites P. chabaudi and P. berghei (23) are not amplified during gametogenesis. In P. berghei and P.falciparum, two different I8S-like rRNAs are present, encoded by two unlinked rRNA genes identified as A and C genes (26, 65). These genes in P. berghei contribute differently to the production of smaIl ribosomal subunits in different developmental stages of the parasite's life cycle. In blood-stage parasites (containing a large majority of asexual forms), the small rRNA is transcribed mainly from the A gene, while in the mosquito stages (oocysts and sporozoites), it is transcribed mainly from the C gene (38). The switch from A gene transcription in the blood stages to C gene transcription in the mosquito stages has been studied in P. Jalciparum (lQ2). Gametocytes contained large amounts of the blood stage-specific A gene transcript; they also contained a long, presumably precursor, form of the mosquito stage-specific C gene transcript carrying an additional 1800 nucleo tides at its 5' end. Following gametogenesis, increasing amounts of the mature C gene transcripts appeared while the A gene transcripts were cleaved at their 3' ends, presumably prior to their complete degradation. To our knowledge this report is the first of replacement of one ribosomal population by another during the transition between two developmental stages. Nucleus, Chromosomes, and DNA Content The nuclei of male and female gametocytes are morphologically distinct when viewed with both light and electron microscopy. That of the female is relatively compact and dense compared to the larger nucleus of the male gametocyte. Condensed chromatin has not been observed in gametocytes or any other stage of the parasite's life cycle. During male gamete formation, however, division of the chromosome material is recognizable by the forma tion of centriolar structures attached to intranuclear spindles (81). Shortly



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after fertillzation, the zygote genome undergoes a meiotic division (82, 83) and the haploid content of the genome is restored prior to sporozoite forma tion. PFGE analysis has determined that the haploid number of chromosomes in P. Jalciparum is 14 (100). Early studies using mitomycin C as an inhibitor of DNA synthesis indicated that DNA was replicated during the early stages of maturation of the gameto cytes (84, 91), although later studies suggested that data from the use of mitomycin C may be unreliable as a test for DNA synthesis in Plasmodium (48). Cytofluorometric analysis of the DNA content of gametocytes in P. berghei (49) and in P. Jalciparum (47) nevertheless showed that in early developing gametocytes the DNA content increases significantly and in P. Jalciparum reaches about twice the haploid amount. The observation of a nuclear spindle in the mature female gametocyte suggested that DNA replica tion may take place at this stage (80). Alternatively, the increase in DNA content may result from DNA amplification (47, 49). Prior to activation, the DNA content of the male gametocytes was little more than the haploid amount. Within a few minutes of initiation of game togenesis, however, cytofluorometric analysis of the male gametocytes showed that their DNA content had increased eightfold (50). DNA replication in male gametocytes is clearly a tightly controlled process and procee:ds with great speed. It is initiated at the time of gamete formation during the process of exflagellation. Characterization and control of the enzymes involved and identification of the origins of DNA replication will be of considerable interest. Expression of Sexual Stage-Specific Proteins Both asexual- and sexual-stage malaria parasites are characterized by the expression of stage-specific proteins. Most studied among the sexual-stage parasites are membrane-bound proteins on the surface of the extracellular forms-the gametes, zygotes, and ookinetes. Several of these proteins are targets of antibodies that can prevent infection in the mosquito when ingested together with the parasites in a blood meal (18). Such proteins could, in principle, form the basis of a malaria transmission-blocking vaccine. In P. Jalciparum, two gamete surface proteins have been described that are synthesized during gametocyte maturation in the blood stream. One has an apparent Mr of 230 kd and the other occurs as a doublet of proteins of apparent Mr of 48 and 45 kd (18). During immunoprecipitation from gametocytes extracted in nonionic detergent, the 230-kd and 48/45-kd proteins remain associated, suggesting that they may form a complex on the gamete surface (59). Antibodies against these proteins prevent fertilization by the parasites in the mosquito midgut. A 25-kd protein has been identified on the surface of the female gamete,


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zygote, and ookinete of P. jalciparum (97). This protein is barely detectable (97) and its gene transcript undetectable (53) in gametocytes; its synthesis begins, however, within minutes of initiation of gametogenesis. Antibodies against the 25-kd protein do not prevent fertilization but stop subsequent development of the parasite in the mosquito, possibly by blocking passage of the ookinete across the mosquito midgut wall (96). While antibodies against the 230- and 48/45-kd gamete surface proteins are readily detected in the sera of many people exposed to malarial infection, none appear to be formed against the 25-kd zygote surface protein (17), probably because of its virtual absence from gametocytes. Targets of transmission-blocking antibodies have been described in other species of Plasmodium including P. vivax (27, 74), P. berghei (106), and P. gallinaceum (56). In P. vivax, one such target is a protein of 23 kd present in the parasitophorous vacuole of both asexual stages and gametocytes and on the surface of the extracellular female gametes (27). One study reported that antibodies that blocked infectivity of P. vivax at high concentration enhanced infectivity at low concentrations (75). Low concentrations of antigamete antibodies may promote agglutination of gametes, leading to improved fertilization efficiency. Among these proteins, the genes coding for the 25-kd ookinete surface protein of P. jalciparum, and its equivalent in P. gallinaceum, have been cloned and sequenced (53, 54) as has the gene for the 23-kd parasitophorous vacuole and gamete surface protein of P. vivax (27). The 25-kd ookinete protein is rich in cystein, which is distributed in four similar domains in the molecule (53). The organization of the cysteins in each domain is similar to that in epidermal growth factor (EGF) (31) and related proteins. Some of these proteins, including lin-12 of Caenorhabditis elegans (37) and the Notch gene product of Drosophila (105), are believed to be involved in cell-cell interactions governing cell differentiation, suggesting that the EOF-like do mains may be involved in these events. The function of the EOF-like domains in the 25-kd ookinete surface protein is unknown, but the domain may be involved in the interactions between the migrating ookinete and the cells of the mosquito midgut wall. The function of these domains may also be to protect the parasite from digestive processes that work on the mosquito blood meal. EOF has been shown to protect cells against attack by acid secretions in mammalian gut tissues (29, 58, 72). As already discussed, a sexual stage-specific protein, Pfg 27/25, has been described in P. jalciparum that is synthesized in gametocytes at a very early stage in their development, between 30 and 40 hours after invasion of a red blood cell (16). The protein is expressed in two apparent Mr of 27 and 25 kd and is extremely abundant, representing about five percent of the total protein of the mature gametocytes. It probably undergoes some form of post-


445

transcriptional modification as peptides exhibit multiple isoelectric points. The exact location and function of the protein are unknown; the gene coding the proteilll has recently been cloned and sequenced (P. Alano & R. Carter, unpublished results). The sequence has no obvious outstanding features or relationships with other known proteins.


CONCLUSIONS

Cellular and molecular studies show that cell specialization during the de velopment of the sexual stages of malaria parasites involves the modification of basic cellular components such as ribosomes and the cytoskeleton into forms specific to these stages. The differentiation processes involve the expression of, no doubt, numerous sexual stage-specific genes, including those coding for ribosomal subunits and tubulin and actin proteins. In P. Jalciparum, one can classify some of the genes coding sexual stage-specific proteins according to the order in which they are expressed, from the early expressiolll of the 27/25-kd protein to the 230- and 48/45-kd proteins ex pressed illl the maturing gametocyte to the 25-kd zygote surface protein expressed predominantly after the initiation of gametogenesis. It is hoped that molecular studies on the expression of such genes, using the mutants with diminished or aberrant capacities for gametocyte production, will provide insights illlto the control and expression of sexual differentiation in malaria parasites. Literature Cited 1. Adler., S., Tchernomoretz, I. 1 943. The extra-I�rythrocytic origin of gametocytes of Plasmodium gallinaceum Brumpt 1 935. Ann. Trop. Med. Parasitol., 37: 148-5 1 2. Aikawa, M. 1988. Fine structure of malaria parasites in different stages of development. See Ref. 102a, pp. 971 30 3. Aikawa, M., Carter, R., Ito, Y., Nijh out, M. M. 1984 . New observations on gametogenesis, fertilization and zygote transfiormation in Plasmodium galli naceum. J. Protozool. 3 1 :403- 1 3 4 . Aikawa, M., Huff, C. G., Sprinz, H. 1 969. Comparative fine structure study of the gametocytes of avian, reptilian and mammalian malarial parasites. J. Ultrastruct. Res. 26:3 1 6-3 1 5. Birago, c., Bucci, A., Dore, E., Fronta Ii, C., Zenobi , P. 1982. Mosquito in fectivity is directly related to the propor tion of repetitive DNA in P. berghei. Mol. Biochem. Parasitol. 6: 1- 12

6. Bishop, A. 1955 . Problems concerned with gametogenesis in Haemospor idiidea with particular reference to the genus Plasmodium. Parasitology 45: 163-85 7. Bishop, A. 1958. An analysis of the development of resistance to metachlo ridine in clones of P. gallinaceum. Parasitology 48:2 1 0-34 8. Bishop, A., McConnachie, E. W. 1 960. Further observations on the in vitro de velopment of the gametocytes of Plas modium gallinaceum. Parasitology 50: 43 1-38 9. Boyd, M. F. 1935. On the schizogonous cycle of P. vivax. Grassi and Feletti. Am. J. Trop. Med. 1 5:605-30 1 0. Breindl, V. 1 926. Studie 0 Plasmodiich. Tr. Ceske Akad. 35: 1-47 1 1. Brockelman, C. R. 1979. Induction of

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