Life cycle of P. berghei

• Differences between different isolates and laboratory lines of P. berghei
• Sporozoites and pre-erythrocytic development
• Erythrocytic (blood stage) development
• Fertilization and zygote development in the mosquito
• Oocyst and sporozoite development
• References

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Differences between different isolates and laboratory lines of P. berghei

In our laboratory we mainly use parasites of the ANKA isolate of P. berghei and many characteristics of the life cycle described below are based on our observations of these parasites, during their growth in Swiss mice or Wistar rats and transmission by Anopheles stephensi. The different isolates (strains) of P. berghei show many similar and stable characteristics of their life cycles. For example, all life cycle stages of the different isolates have a similar morphology and duration of development. No variation in iso-enzymes has been found and they show in general a comparable sensitivity to antimalarial drugs and other inhibitors.
There are however (small) differences between P. berghei parasites of different isolates and laboratory lines that influence different characteristics of infection such as the course and intensity of blood infections, gametocyte production, virulence and pathology (see table 1 for definition of the terms isolate, line and clone; isolates are often referred to as strains in rodent parasites).
Reported variations are mainly the result of variation in environmental factors and by differences in vertebrate or mosquito hosts used in different studies, but may also result from (genetic) differences between the isolates and laboratory lines of P. berghei. A number of well-documented variable characteristics of P. berghei are listed below. These characteristics have been largely described at the level of phenotype and, therefore, their genetic and molecular basis are unknown (see also table 2).

1. Reticulocyte preference. P. berghei has a strong preference for invading reticulocytes. To our knowledge no lines of a single isolate of P. berghei have been obtained in the laboratory that show stable, significant differences in preference for invading different red cell populations. There is however evidence that different isolates show 'small' but significant differences in reticulocyte preference. For example the NK65 isolate has a stronger preference for reticulocytes in comparison to the ANKA isolate in Swiss mice (our own observations; ref. #1). The reticulocyte preference can also be host-dependent: we observed that parasites of the ANKA isolate have a stronger reticulocyte preference in Wistar rats than in Swiss mice.
2. Number of merozoites per erythrocytic schizont. In general parasites growing in more mature erythrocytes produce  less merozoites (6-12 merozoites per schizont) then those that grow in reticulocytes (12-18 merozoites per schizont) (own observations, ref. #2).
     
3. Sequestration of erythrocytic schizonts. In most laboratory animals the erythrocytic schizonts disappear from the blood circulation and sequester in the capillaries of the inner organs, such as the spleen, lungs and adipose tissue. To our knowledge no stable lines of P. berghei exist which show significant differences in the site or level of sequestration of their erythrocytic schizonts. However, small differences might exist between different lines in the number of mature schizonts circulating in the peripheral blood. We have evidence that lines of the K173 isolate, which have been mechanically passaged for many years in laboratory rodents have more schizonts in the peripheral blood then lines of the ANKA isolate. The site of sequestration of the erythrocytic appears to be influenced by host factors. For example, schizonts of the ANKA strain in young (BALB/c x C57BL/6) mice sequester in the microvasculature of the brain (ref. #3), whereas in many other rodent hosts no significant sequestration of parasitised erythrocytes in the brain has been observed. Schizonts of P. berghei preferentially sequester in the spleen, lungs and adipose tissue.
4. Virulence and pathology. In many strains of laboratory mice and rats an infection with P. berghei causes death within 1 to 3 weeks and many animals show cerebral complications. A large number of environmental factors influence the virulence and pathology of P. berghei and as implied above the (genetic background of the) host is one of the most important factors. In most laboratory rodents P. berghei causes rapidly fulminating infections leading to death, whereas in the natural host it causes long lasting, chronic infections with low parasite densities. In many mouse strains P. berghei causes cerebral complications, but between rodent strains significant differences exist in the susceptibility to cerebral complications. For example, older laboratory rats show no cerebral complications and are able to clear parasites from the blood and recover from a P. berghei infection (ref. #4). The use of KO mice- deficient in production of certain cytokines or deficient in expression of adhesion molecules on host endothelia, has emphasised the importance of host-genetic factors in virulence and pathology caused by P. berghei (ref. #5-8). In addition to the genetic background other host factors such as age and diet (ref. #9) of the host influences the course of infection, virulence and pathology. Additional environmental factors that influence infection characteristics are the route of infection (sporozoite induced versus blood infections) and the dose of infection. In addition to the host and other environmental factors, (genetic) differences between laboratory lines of P. berghei can affect virulence and pathology. Several laboratory lines of P. berghei have been produced that demonstrate a lower virulence. For example, a stable attenuated parasite line (XAT) of the NK65 isolate, derived by irradiation, shows a low self-resolving parasitemia in mice (ref. #10, 57). Also from the K173 isolate a less virulent line has been selected (ref. #11).
5. Gametocyte production. The gametocyte production, defined as the percentage of blood stage parasites that differentiate into gametocytes, is influenced by environmental factors (own observations; ref. #12; see also below). In addition, stable, genetic differences exist in gametocyte production between laboratory lines of P. berghei (ref. #13).
6. Sex ratio of gametocytes.  In the rodent parasite P. vinckei the sex ratio is influenced by environmental factors (ref. #14). We found evidence that the sex ratio of P. berghei gametocytes might be affected by (unknown) environmental factors (unpublished observations; see below).
7. Gametocyte infectivity.  Infectivity of gametocytes to mosquitoes, as determined by the number of oocysts formed, is affected by a number of blood factors (both immune and non-immune serum factors)(refs. #15,16). In general, infectivity of P. berghei gametocytes drops during the later stages of blood stage infections in laboratory rodents (see also below).
     
8. Intensity of mosquito infection. The intensity of mosquito infection, defined as the number of oocysts, is dependent on the gametocyte infectivity (see above) and the susceptibility of the different mosquito hosts used in the laboratory (refs. #2,4). Between different Anopheles species and between strains (lines) of one species large (genetic) differences have been reported in the susceptibility to support oocyst infection (see below).
9. Duration of development in the mosquito (sporogony).  The length of the sporogonic cycle in the mosquito is dependent on the environmental temperature (ref. #2).
10. Salivary invasion of sporozoites. The rate of salivary gland invasion is different in the various mosquito hosts that are used in the laboratory. For example, the sporozoite invasion rate is higher in A. stephensi then in A. quadrimaculatus (ref. #4)
11. Infection rate of liver cells by sporozoites. The infection rate of sporozoites in liver cells is different in the different species and strains of laboratory rodents. These rodents range from almost totally refractory to 'highly' susceptible to the development of the pre-erythrocytic stages (ref. #4). This level of susceptibility is not only determined by the sporozoite infection rate but can also be influenced by innate immune responses against developing sporozoites, just after invasion of the hepatocyte (ref. #20)
12. Size of pre-erythrocytic schizonts. The size and the number of merozoites in mature pre-erythrocytic schizonts is reported to be different in different host species (see references in ref. #2)
13. Chromosome size. See for a description of chromosome size variation Genome of P. berghei

Table 1: Terms and definitions used for the description of malaria parasites in genetic and genome research (from refs. #17,18,19)

Isolate (strain)	A sample of parasites taken from an infected person or animal on a unique occasion; an isolate is uncloned, and thus may contain more than one genetically distinct parasite clone (in rodent parasites isolates are often referred to as strains).
Line	Parasites of a single species derived from a single isolate, not necessarily cloned, which have some common phenotype, e.g. drug-resistance.
Clone	The progeny of a single parasite, normally obtained by manipulation or serial dilution of uncloned parasites and then maintained in the laboratory. All the members of a clone have been classically defined as genetically identical, but this is not necessarily the case, since members of the clone may undergo mutations, chromosomal rearrangements, etc, which may survive in the culture conditions.

Table 2: A number of characteristics of P. berghei that are variable either as a result of genetic differences between isolates and laboratory lines or as a result of variable environmental factors (see text for more details).

	Variation influenced (caused) by:
Variation in:	Genetic differences between parasites	Genetic background of host	Other environmental differences
Reticulocyte preference	+(?)	+	?
No. of merozoites/schizont			Age of host erythrocyte
Sequestration of schizonts:
Site:		+
Level:	+(?)
Virulence/Pathology	+	+	Age of host; Host diet; Artificial infection methods
Gametocyte production	+		+ (unknown blood factors)
Sex ratio			+ (?; unknown blood factors)
Gametocyte infectivity			Blood factors
Intensity mosquito infection (oocyst number)		+	Blood factors
Sporozoite invasion: salivary glands		+
Sporozoite invasion: liver cells		+
Size of pre-erythrocytic schizont (number of merozoites)		+

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Sporozoites and pre-erythrocytic development

An infection starts with the bite of an infected mosquito which inoculates the haploid sporozoites in the blood stream of the vertebrate host.

The (ultra structural) morphology of sporozoites of P. berghei resembles that of sporozoites of human parasites.
Sporozoites home to the liver and invade hepatocytes. Intact sporozoites can be observed inside hepatocytes from a few minutes to several hours after inoculation. Two major surface proteins of sporozoites, CS and TRAP (others may yet be discovered) which play a role in the journey to and invasion of hepatocytes, are conserved between rodent and human malarias. Molecular studies on surface proteins of sporozoites of P. berghei have provided insight into the gliding motility and cell invasion of the sporozoite (ref. #21). Hepatocyte invasion is mediated by invagination of the host cell plasma membrane to form a parasitophorous vacuole which surrounds the invading sporozoite. Sporozoites can migrate through several host cells before invading a hepatocyte by formation of a parasitophorous vacuole (ref. #59) Within the hepatocyte the sporozoite develops within 47-52 hour via the trophozoite stage into the mature schizont, that can contain 1500-8000 merozoites (the total number of merozoites per mature schizont can vary in different hosts). A detailed description of (the ultra-structural) morphology and development of liverstages of the ANKA strain of P. berghei in brown-norway rats is found in refs. #60-62. In brief: Within 51h the sporozoite with a length of 12µm develops into a mature liver schizont with a diameter of 30µm. The merozoites formed have a length of 1.6µm, comparable with the size of erythrocytic merozoites. Mature liverschizonts contain about 8000 merozoites. Nuclear division starts around 24 after invasion of the hepatocytes, which means that at least 13 nuclear divisions occur within a period of 26 hour. The basic pattern of merozoite formation is comparable to that observed in the erythrocytic schizonts and during sporogonic multiplication in the mosquitoes.
The molecular events controlling exo-erthrocytic development of mammalian malaria parasites are largely not understood (ref. #22), despite a rather detailed knowledge on the immunological responses against these stages in rodent hosts (ref. #23). After rupture of the liver cell the merozoites of the pre-erythrocytic schizont are released into the blood stream where they invade red blood cells.
In P. berghei there is no evidence for the hypnozoite stage in the liver which is found in P. vivax and some other non-human primate malarias and is a 'dormant' (arrested) liver stage. The hypnozoite stage can persist for prolonged periods before it starts to develop into a liver schizont, resulting in a normal blood infection (ref. #25). However, evidence has been presented for the existence of slow developing ('chronic') liver schizonts of rodent parasites in the natural host (ref. #24).

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Erythrocytic (blood stage) development

The haploid merozoites that are released from the liver schizonts, invade red blood cells.
P. berghei has a preference for reticulocytes but can also invade mature red blood cells (small differences in reticulocyte preference can be observed between different isolates and laboratory lines of P. berghei and in different host strains and species).

   

Asexual development See Morphology P. berghei LM for light-microscope pictures of the different life cycle stages.
Within an erythrocyte the merozoite develops into a trophozoite, characterized by an increase in cell size and cytoplasm. The trophozoite consumes the haemoglobin of the red blood cell, thereby producing crystals of the brown haemozoin that can be observed as the characteristic pigment granules in the cytoplasm. The development of the merozoite (via the ringform) into a mature trophozoite, just before nuclear division starts, takes around 16 hours. Towards the end of the trophozoite stage, the parasite duplicates its DNA. Replication of the DNA is followed by nuclear division, leading to a binuclear parasite. With this first nuclear division, the parasite enters the schizont stage. During schizogony, which takes 6-8 hours, the parasite replicates its DNA and divides its nuclei a number of times (ref. #26), forming a syncytial cell with 8-24 nuclei. Nuclear division is endomitotic, a common feature in unicellular eukaryotes, and the segregating chromosomes and the spindle apparatus remain within the nuclear envelope throughout the process (ref. #27). The individual chromosomes do not condense into tight, visible structures like those seen in classical mitosis. Only towards the end of schizogony does the parasite start to divide its cytoplasm by budding of small, uninuclear merozoites.
The (ultra structural) morphology of merozoites of P. berghei resembles that of merozoites of human parasites (see ref. #27 for a review on the ultra structure of Plasmodium falciparum asexual blood stages). The merozoite has many special features related to invasion of red blood cells, such as the apical organelles, i.e. the rhoptries, micronemes and dense granules. A number of (surface) proteins of merozoites, which are likely to play a role in invasion, appear to be conserved between rodent and human malaria parasites (ref. #28,29). Examples are MSP1, MSP4/5, AMA1, MAEBL, RhopH3 and SUB2. Like P. falciparum, P. berghei has a plastid like organelle that contains a circular, extra-chromosomal genome of 30.7 kb. This DNA shows 70-95% homology with the 35kb circle of P. falciparum and arrangement of characterised genes is similar to that found on the P. falciparum 35-kb circle (ref. #30). In addition, rodent parasites contain a 6kb extra-chromosomal mitochondrial DNA, homologous to the mitochondrial genome of P. falciparum (refs #31,32). The total duration of the asexual blood stage development is 22-24 hours. Mature schizonts in mature erythrocytes usually contain less merozoites (8-12) in comparison with schizonts in reticulocytes (16-18 nuclei). In the mature schizonts, the pigment granules become compacted in the pigment vacuole into a single, dense, rounded mass.
Immature and mature schizonts disappear from the peripheral circulation and sequester in the capillaries of inner organs, such as the spleen and lungs (differences in the level and site of sequestration can be observed between different isolates and laboratory lines of P. berghei and between different host strains and species). In rodent malarias the proteins on the surface of infected erythrocytes and their role in sequestration are largely unknown. No proteins have so far been described that are equivalent to the variant surface proteins of P. falciparum infected cells, such as members of the PfEMP. STEVOR and RIFIN family, although recently a extensive gene family has been described in rodent parasites (ref. #64) and in the human parasite P. vivax (ref. #63) encoding variant antigens that are most likely exported to the surface of infected erythrocytes (PIR's = Plasmodium interspersed repeats). See ref. #34 for a review on P. falciparum proteins on infected erythrocytes and their possible role in sequestration. After rupture of the schizonts, the free merozoites invade new red blood cells, resulting in an increase in the parasitemia (= percentage of infected red blood cells).
The blood stage development of P. berghei in laboratory rodents is usually asynchronous, that means that the different developmental stages, such as rings, trophozoites and schizonts are simultaneously present in the blood during the course of infection.

  

Sexual development. See Morphology P. berghei LM and Morphology P. berghei EM for pictures (light microscope and electron microscope) of the different life cycle stages.
In each asexual cycle a small proportion of parasites stop asexual multiplication and differentiate into sexual cells, the so-called gametocytes. These haploid macrogametocytes (females) and microgametocytes (males) are the precursor cells of the female and male gametes.
We have evidence that in each blood stage cycle 5-25% of the parasites is committed to sexual differentiation (ref. #12). This relatively fixed percentage of cell that differentiates into sexual cells is different from the sexual development in P. falciparum where periods of 'pure' asexual multiplication are alternated with waves of gametocyte production.
In P. berghei, the merozoites of liver schizonts are able to differentiate directly into gametocytes after invasion of the erythrocyte (ref. #35).
The period of the development of a merozoite into a mature gametocyte is 'short' and takes only 26-30 hour (ref. #12, 36). During the first 16-18 hours of development the 'gametocytes' cannot be distinguished from asexual trophozoites at the light-microscope and electron-microscope level (ref. #12). After 18-22 hour sex-specific features develop, such as a single enlarged nucleus, even distribution of pigment granules throughout the cytoplasm and size of the cells (filling the red blood cell). At the EM level osmiophilic bodies can be found. However, females cannot yet be distinguished from male gametocytes. Only after 24h do male specific features become apparent, such as an enlarged, eccentric nucleus, less dense stained cytoplasm as a result of breakdown of ribosomes and less osmiophilic bodies compared to female gametocytes (EM level). The developmental time of 26-30 hour is short in comparison with the developmental time of 8-11 days of gametocytes of P. falciparum. Also the morphology of the gametocytes of these two species is different: banana-shaped gametocytes in P. falciparum and round to oval gametocytes in P. berghei. The morphology, developmental time and production of gametocytes in P. berghei show more similarity with those of gametocytes of the other human and non-human primate malarias.
It is not known when commitment to sexual differentiation takes place in P. berghei parasites. There is some evidence that sexual commitment in P. berghei occurs in the trophozoite stage between 12 and 16 hours after invasion (ref. #12), directing the differentiation of these trophozoites into gametocytes. Again this would be different from P. falciparum where commitment to sexual differentiation occurs in the previous cycle prior to schizont maturation. Therefore, the merozoites of P. falciparum are already committed to become a gametocyte before invading a new red blood cell (refs. #37-39), while in P. berghei committment probably occurs after invasion (although from the above, committment in both species seems to occur in the trophozoite).
The molecular mechanisms that induce and regulate the switch from asexual multiplication to sexual differentiation are unknown. Both in P. falciparum and in P. berghei there is evidence that environmental factors influence the switching mechanism (refs. #12,38). Recently evidence has been presented that erythropoietin levels can influence the ratio of male and female gametocytes in the rodent parasite P. vinckei (ref. #40). Also in our laboratory evidence was found that the male/female ratio of P. berghei is under influence of environmental factors in the blood (a higher male:female ratio in phenylhydrazine treated rodents; unpublished observations).
We have no evidence that developing gametocytes of P. berghei sequester in capillaries of inner organs during development, like the gametocytes of P. falciparum. Based on light-microscopic observations on the morphology of gametocytes of rodent parasites, it has been proposed that certain stages of gametocytes sequester preferentially in blood capillaries from which mosquitoes take their blood meal (ref. #24). In our laboratory we could not demonstrate differences in infectivity of gametocytes taken up by mosquitoes and gametocytes obtained from tail or heart blood of infected rodents on which the mosquitoes had been fed (ref. #41).

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Fertilization and zygote development in the mosquito

When a mosquito feeds on an infected host, only the mature gametocytes can undergo further development in the mosquito midgut. This involves an active escape of the gametocytes from the red blood cell and the formation of gametes (the process of gametogenesis). The female gametocyte differentiates into a single, spherical female gamete (macrogamete) whereas the male gametocyte produces 8 'sperm-like' gametes. The formation of the male gametes is a rapid process. Within 10 minutes, three rounds of DNA replication (ref. #42), nuclear division and the formation of 8 flagellar axonemes occur, resulting in the production of eight, motile gametes. Three environmental triggers have been described that induce the differentiation of the gametocytes into the gametes: a drop in temperature of the infected blood to at least 5°C below that of the vertebrate host, a rise in pH from 7.3 to 7.8-8.0 and the presence of a gametocyte activating factors (GAF)(refs. # 43-45) In P. berghei the mosquito-derived GAF is xanthurenic acid (ref. #45). In contrast to the distinct formation and morphology between the gametocytes of P. berghei and those of P. falciparum, the process of gamete formation and gamete morphology are highly comparable in these species. It has been demonstrated that a number of surface proteins of the gametes are conserved between rodent and human parasites. For example, a high level of conservation exists in structure of members of the P48/45 protein family, such as P48/45, P47 and P230 (refs. #46, 47). Fertilisation takes place by penetration of the haploid male gamete into the haploid female gametocyte, resulting in the diploid zygote. Between 10 min. and 1 hour after the formation of gametes, fertilisation and fusion of the male and female nuclei takes place (ref. # 42) followed by meiosis (ref. #48). Meiosis is not directly followed by nuclear division, resulting in single nucleated zygote/ookinete with 2-4 times the haploid amount of DNA (ref. # 42). The spherical zygote develops into a banana-shaped, motile ookinete within a period of 18-24 hour. The small pigment granules that are scattered throughout the cytoplasm of the gametocytes/zygotes become 'packaged' into a few clusters in the mature ookinete. Ookinetes of human and rodent parasites show a comparable morphology and like other invasive stages of Plasmodium (merozoites, sporozoites) contain an apical complex (ref. #49) for penetration and traversing of cells of the midgut epithelium. Several (surface) proteins of ookinetes that are involved in interaction of the ookinete with the mosquito midgut such as the peritrophic matrix, midgut epithelium and basal lamina, are conserved in structure between rodent and human parasites. Examples are Chitinase, CTRP, P25 and P28 proteins (refs. #50-52).

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Oocyst and sporozoite development

Mature, motile ookinetes traverse the midgut epithelium by invasion of cells of the epithelium and settles between the basement cell membrane and the basal lamina of the midgut wall (refs. #53,54). Most probably the P. berghei ookinete does not invade a specific midgut cell type and ookinetes traverse serially the cytoplasm of several midgut cells before entering and migrating through the basolateral intercellular space to access the basal lamina. The invaded cells commit apoptosis (ref. #58, #65). Upon emerging from the epithelial cell, the ookinete makes contact with, but appears to be unable to penetrate the basal lamina. Here the parasites rapidly round up and develop into the oocyst stage. After a growth phase of the oocyst asexual, mitotic replication results in the formation of a mature oocyst that contains thousands of daughter cells (sporozoites). The oocysts increase in size from 2-3µm in diameter to about 40µm within 10-13 days. Many features of infection characteristics in the mosquito, such as numbers of oocysts produced and numbers of sporozoites found in the salivary glands is dependent on the Anopheles species, that is used to transmit P. berghei. In Anopheles stephensi infected with the ANKA strain of P. berghei an average of 8000 sporozoites per oocyst have been recorded (ref. #15). Oocyst rupture and the haploid sporozoites are released into the haemocoele that will invade the salivary glands. It has been found that only 2% of the oocysts sporozoites reach the glands in the P. berghei/A. stephensi combination (ref. #15). The first sporozoites reach the salivary gland at day 13-14 after the infectious blood meal. Sporozoites migrate through cells of the gland and exit into the extracellular secretory space where the sporozoites can persist for many weeks before being injected into a new host. An average number of 11.000 salivary gland sporozoites have been recovered per A. stephensi mosquito over a period of twenty years of infecting mosquitoes (ref.#15). Per bite probably only 20-50 sporozoites are delivered to the host (ref. #15). Rodent parasites are extensively used to provide insight into parasite-mosquito interactions. For a review on immune responses in mosquitoes against P. berghei see ref. #55. For a review on interactions of the ookinete with the midgut epithelium and basal lamina see ref. #53. For a review on the interaction of sporozoites and the salivary gland see refs. #21,56.

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