1.4 Parasites and vectors - Life-cycle of Plasmodium species

Question 1

Aims

Answer 1

To provide a detailed, stage-by-stage consideration of the life-cycles of human malaria parasites.

Question 2

Objectives

Answer 2

After working through this session, you should be able to: Describe the life-cycles of the human malaria parasites.Relate stages in the life-cycles to the causes of disease.Explain the processes of cell invasion and intracellular development during the life-cycle.Provide reasons why some of the molecules involved in cell invasion might be candidate targets for vaccines.

Question 3

Introduction

Answer 3

This session is a detailed, stage-by-stage consideration of the life-cycles of human malaria parasites. Knowledge of the life-cycle is important for understanding the disease, the treatment, the development of drugs and vaccines, andcontrol. Much of what we know about the details of the life-cycles of the five human malaria parasites has been derived from studies on other species. This is not a problem because, as we have already seen, the life-cycles of allPlasmodiumspecies in mammals are basically the same and differ only in detail (the importance of animal models was discussed in Session 1.3Parasites and vectors:Plasmodiumspecies and their use as animal models.

Question 4

Question 1 Infection begins when infectiveXXXstages injected by a femaleAnophelesmosquito enter the bloodstream. Within 30-60 minutes, sporozoites invade liverXXX, round up and then begin to divide for 5-15 days depending on the species. This phase of multiplication is theXXXstage and results in the formation of a multinucleate exoerythrocytic schizont containing 10–30,000 uninucleate cells.

Answer 4

Infection begins when infectivesporozoitestages injected by a femaleAnophelesmosquito enter the bloodstream. Within 30-60 minutes, sporozoites invade liverhepatocytes, round up and then begin to divide for 5-15 days depending on the species. This phase of multiplication is theexoerythrocyticstage and results in the formation of a multinucleate exoerythrocytic schizont containing 10–30,000 uninucleate cells.

Question 5

Question 2 The uninucleated cells mature to formXXXthat flood into the bloodstream. The merozoites within one to two minutes attach to and enter red blood cells (erythrocytes) and begin theXXXstage of the cycle. Inside a parasitophorous vacuole, within the red blood cell, a merozoite becomes aXXXor feeding stage. After a while, the nucleus begins to divide resulting in the production of an erythrocytic schizont containing 8-24 merozoites. This asexual reproductive cycle occurs repeatedly and though very rare, it is possible that up to 40% of red blood cells may become infected. Eventually, some merozoites differentiate into sexual forms calledXXX, which cannot develop further until taken up by an appropriate mosquito.

Answer 5

The uninucleated cells mature to formmerozoitesthat flood into the bloodstream. The merozoites within one to two minutes attach to and enter red blood cells (erythrocytes) and begin theerythrocyticstage of the cycle. Inside a parasitophorous vacuole, within the red blood cell, a merozoite becomes atrophozoiteor feeding stage. After a while, the nucleus begins to divide resulting in the production of an erythrocytic schizont containing 8-24 merozoites. This asexual reproductive cycle occurs repeatedly and though very rare, it is possible that up to 40% of red blood cells may become infected. Eventually, some merozoites differentiate into sexual forms calledgametocytes, which cannot develop further until taken up by an appropriate mosquito.

Question 6

Sexual and sporogonic stages

Answer 6

Within the gut of the mosquito, and enclosed within a peritrophic membrane, the final phases of the life-cycle, the sexual and sporogonic stages, occur. The gametocyte-infected erythrocytes rupture to release extracellular male and female gametes. The male fertilises the female, resulting in the formation of a zygote called an ookinete.

Question 7

Sexual and sporogonic stages

Answer 7

The ookinete burrows through the midgut wall, encysts on the outer surface and becomes an oocyst within which there is a phase of multiplication resulting in the formation of large numbers of sporozoites. Rupture of the oocyst releases the sporozoites which migrate to the salivary glands ready to initiate a new infection in humans when the mosquito takes another blood meal.

Question 8

Sexual and sporogonic stages Gametogenesis

Answer 8

The fertilisation of the female gamete (macrogametocyte) by the male gamete (microgametocyte) results in an ookinete which becomes an oocyst in the midgut wall of the mosquito.

Question 9

Sexual and sporogonic stages

Answer 9

Sporozoites leave the bursting oocyst (attached to the mosquito’s stomach wall), enter the haemocoel then enter the mosquito’s salivary glands and initiate a new infection the next time it feeds.

Question 10

Sexual and sporogonic stages

Answer 10

The life-cycle of the malaria parasite is thus very complex and consists of: the infective stage or sporozoite,two phases of asexual reproduction, one in the liver and the other, which is repeated several times, in the blood, anda sexual stage that begins in the blood and is completed in a mosquito in which sporozoites are formed.

Question 11

Sexual and sporogonic stages

Answer 11

The various stages in the life-cycle are structurally, biochemically and immunologically different and a knowledge of these stages is essential if you want to understand the disease processes, how drugs work, the prospects for vaccination and the control of the disease. This information will be accumulated progressively as you work through the module.

Question 12

Sexual and sporogonic stages

Answer 12

The life-cycle of a typical mammalian malaria parasite is shown. It has two parts: in the human host and in the mosquito (Anopheles) host. In humans, there are two parts, the exoerythrocytic cycle in the liver followed by the erythrocytic cycle in the blood. The exoerythrocytic cycle starts when a sporozoite penetrates a liver celland merozoites grow in the liver cell. The erythrocytic cycle starts when the liver cell bursts and releases merozoites which penetrate the red blood cells. A schizont forms and ruptures, releasing merozoites. Some of the merozoites reinvade red blood cells, other merozoites evolve into microgametocytes and macrogametocytes. The gametocytes are taken into the mosquito’s stomach with a blood meal, starting the mosquito part of the parasite life-cycle.In the mosquito, the gametocytes undergo gametogenesis and fertilisation to form an ookinete which penetrates the midgut wall of the mosquito and undergoes sporogony: it develops into an oocyst which ruptures and liberates sporozoites which penetrate the salivary gland. The sporozoites are then injected into a human host with the mosquito saliva during a blood meal, which completes the life-cycle.

Question 13

Parasite numbers

Answer 13

An important feature of the life cycle that we shall refer to again as we consider each stage of the life cycle in more detail is how numbers change. The graph’s vertical axis is a log scale. Numbers start low with about 10 liver schizont parasites, peaking a 1012for asexual blood stages in man. This drops to −102 as sexual blood stages develop. This is the stage taken up by mosquitos ookinetes and macrogametes develop and fuse to form oocysts which then develop and multiply to 104 sporozoites. These migrate to the salivary glands where about 10 sporozoites are inoculated into a host during a blood meal.

Question 14

Parasite numbers

Answer 14

The most dramatic effect occurs when humans are first infected. One sporozoite that successfully invades a liver parenchyma cell begins asexual multiplication and, within a week or so the EE schizont ruptures to release up to 30,000 merozoites. Further increase in numbers occur following invasion of erythrocytes and several cycles of blood-stage schizogony. There is a second period in the life-cycle when numbers increase markedly. In the mosquito, one ookinete gives rise to one oocyst, then within the cyst repeated multiplication results in the release of several thousand sporozoites.

Question 15

Parasite numbers

Answer 15

You should notice also that when fertilisation occurs in the midgut of the mosquito numbers are then very low. We shall now consider the different stages in more detail, and we’ll start by looking more closely at the sporozoites…

Question 16

Sporozoites

Answer 16

“Mosquitoes inject sporozoites into the skin where they remain for periods of up to 15 mins before finding their way to the liver. Recent intravital microscopic studies in rodents have shown that sporozoites are injected into avascular parts of the skin rather than directly into the blood circulation.” Vanderberg JP and Frevert U,International Journal of Parasitology There is also recent evidence that sporozoites induce immune responses in the draining lymph nodes of the skin site (Guilbrideet al. 2012).

Question 17

Sporozoites

Answer 17

Sporozoites are uninucleate, spindle-shaped structures measuring about 10 × 1 µm. At the anterior end of the sporozoite, there is an apical complex consisting of a number of organelles. This complex, which is characteristic of all the apicomplexan (sporozoan) protozoa, is also seen in merozoites and ookinetes and is involved in cell invasion.

Question 18

Sporozoites

Answer 18

From an immunological viewpoint, the most interesting aspect of the sporozoite is a thick surface coat outside the plasma membrane. The coat largely consists of a single protein termed the circumsporozoite protein (CSP).

Question 19

Sporozoites

Answer 19

InP. falciparum, the CSP contains a tandem repeating sequence of the amino acids asparagine-alanine-asparagine-proline (written in single letter code as NANP) in its central region. Antibodies against the repetitive region dominate the antibody response to the CSP.Other species also have tandem repeats but of different sequences of amino acids.

Question 20

Sporozoites

Answer 20

Sporozoites circulate in the blood for up to one hour, cross the sinusoidal barrier in the liver through either the endothelial cells or the Kupffer cells and then enter and infect hepatocytes. Initially, they enter then pass through a few hepatocytes before selecting one and settling there.Another sporozoite surface molecule, thrombospondin-related adhesive protein or TRAP is, like CSP, a vaccine candidate molecule, as we shall see later.

Question 21

Exoerythrocytic stages

Answer 21

The exoerythrocytic (EE) stages have been most intensively studied in laboratory animals and inin vitroculture systems. When the sporozoite enters the host hepatocyte, it becomes enclosed in a parasitophorous vacuole, loses its structural identity and rounds up. The nucleus then begins a normal process of nuclear division. It was once thought that the nucleus simply split up – hence the term schizont, which is derived from the Greek for ‘split’.

Question 22

Exoerythrocytic stages

Answer 22

The number of divisions is genetically controlled and varies from species to species; it affects the time to maturity, the size of the schizont and the number of merozoites produced.

Question 23

Exoerythrocytic stages

Answer 23

In some strains ofP. vivaxandP. ovale, not all the sporozoites are destined to divide immediately; after rounding up, some variously remain dormant for weeks, months or a few years. Such dormant stages are called hypnozoites, from the Greek god of sleep, Hypnos. The hypnozoites have a single nucleus and are very small – less than the diameter of a red blood cell (see the figure below).

Question 24

Exoerythrocytic stages

Answer 24

Liver section stained with a fluorescent anti-malarial antibody. The large stained body is a developing exoerythrocytic schizont; the very small stained body above is a dormant hypnozoite. A large green oval and a much smaller green circle are visible on a background of liver cells stained red-brown with the cell membranes showing as red lines. The large green oval, which is about twice the size of the liver cells, is a developing exoerythrocytic schizont. The small green circle in one other liver cell is a hypnozoite. The hypnozoite is highlighted and labelled.

Question 25

Merozoites

Answer 25

There is only one phase of exoerythrocytic schizogony in mammalian Plasmodium species and for that reason it is also called pre-erythrocytic schizogony. (In contrast, some avian species may have several exoerythrocytic division cycles in the reticuloendothelial system, including the capillary endothelium, and it is even possible for erythrocytic merozoites to initiate infections there).

Answer 26

Maturation of the exoerythrocytic schizont results in the formation of several thousands of merozoites and finally the rupture of the parasitophorous vacuole. Studies with P. berghei in mice, show that this is followed by the release of merozoite-filled vesicles termed merosomes into the liver sinusoids. The merosome membrane is modified to avoid phagocytosis by the abundant Kupffer cells that line liver sinusoids and which readily phagocytose free merozoites. The merosomes then rupture and release their content of merozoites in the bloodstream to facilitate invasion of erythrocytes. A similar scenario is likely in human malaria although this is yet to be confirmed.

Answer 27

The merozoites are lemon-shaped structures measuring about 1.5 × 1 µm that must find, attach to, and enter a red blood cell within a few minutes.

Answer 28

As we progress through this section, we shall make comparisons between the Plasmodium species. In many cases, it will have to be only four species as we still have much less information about P. knowlesi as a human infection, so are not always able to include it in the comparisons.

Question 29

Question 3 Having worked through the table, select one or more features of each of the five species of Plasmodium that determine characteristics of infection with that parasite, that is, five answers, one for each of the parasites.

Answer 29

We cannot guess what answers you will give but that does not matter as the exercise is about taking note of the features of each infection. Some examples are as follows: P. vivax: Relapse infections are a feature of this parasite and these are very important in terms of epidemiology, control and treatment. You might also note that it is a pathogenic infection which is contrary to the old idea that P. vivax is a benign infection. P. ovale: Again there is a hypnozoite stage (causing relapses). It is less pathogenic than P. vivax (but it is still a symptomatic infection as those who have had it will testify). P. malariae: This is the ‘slow’ parasite with a 72 hour erythrocytic cycle and a small number of merozoites produced by each blood schizont. Consequently, the parasitaemia is low. P. falciparum: Can develop most rapidly in the liver, the EE schizonts produce many more merozoites than the other species so the pre-patent and incubation periods are short and the parasitaemias can reach much higher (fatal) levels. P. knowlesi: The notable feature is the quotidian (24 hour) erythrocytic cycle which means the parasitaemia rises rapidly and the infection is very pathogenic and can sometimes be fatal.

Question 30

Question 4 In human malaria, the term 'pre-erythrocytic' (and 'exo-erythrocytic') are used to describe the same phase of the Plasmodium life cycle. Why is this and is it important?

Answer 30

'Pre-erythrocytic' implies that this stage occurs before the blood stages. We now know that there is only one phase of development in the tissues (i.e liver), but in those malarias that have hypnozoites (P. vivax and P. ovale), these tissue stages will be activated after the blood cell stage which is confusing. In some avian malarias, there are exoerythrocytic schizonts produced from blood-stage merozoites or from exoerythrocytic merozoites as well as from sporozoites in the reticuloendothelial cells, suggesting that in these malarias the EE stages may be different. Both terms will continue to be used but we have opted for now to use exo-erythrocytic or EE forms in all cases.

Question 31

Question 5 What do you think might be the evolutionary significance of the dormant hypnozoite stage of P. vivax?

Answer 31

The hypnozoite is a dormant stage that enters its division cycle long after the initial infection. It is thought that this stage is an adaptation to more temperate regions when an infection acquired late in the summer would not be able to complete its development in a mosquito. The later maturation of hypnozoites would give the parasite a chance to infect mosquitoes early the next summer. It also facilitates the continued transmission of these relapsing malarias when the suitable season for transmission is only a matter of weeks, in very northerly or very dry areas. The function of the hypnozoite in a temperate climate with limited transmission season due to long cold winters, when mosquito numbers are very low, is paralleled in more tropical regions where there may be a limited transmission season because of the long dry season.

Question 32

Question 6 What is meant by the terms 'recrudescence' and 'relapse'?

Answer 32

Relapses are parasitaemias that result from the maturation of hypnozoites long after the first waves of parasitaemia have disappeared. Recrudescences are parasitaemias that arise from continuing low-level erythrocytic infections. Only an exoerythrocytic form can give rise to a relapse and only an erythrocytic form can give rise to a recrudescence.

Question 33

Question 7 The multiplication seen during the exoerythrocytic stage of P. malariae produces 15,000 merozoites from a single sporozoite after 14 days. Do you think this is a rapid rate of multiplication?

Answer 33

No

Question 34

Question 8 Assuming that the parasite divides first into two and then into four and so on, calculate how many cycles of division it would require to produce 15,000 merozoites a) 4-5, b) 7-9, c) 11-12, d) 13-14

Answer 34

d) 13-14 - It would take 13 divisions to give rise to 8,192 ( 213=8192) merozoites, 14 divisions to give rise to 16,384 (214= 16,345) so the answer lies between 13 and 14.

Question 35

Question 9 Now consider the number of days from infection to the maturation of the schizont and the release of merozoites. What is the approximate rate of division in P. malariae? One division every a) hour b) 12 hours c) 24 hours d) 48 hours

Answer 35

P.malariae takes 14 days to produce about 15,000 merozoites, equivalent to about one division every 24 hours.

Question 36

Question 10 Now do the same calculations for P. vivax, P. ovale and P. falciparum.

Answer 36

What you will notice is that P. ovale and  P.malariae have similar rates of division , whereas the rate of division for P. vivax and P. falciparum is much faster, with about one division every 12 hours.  Unfortunately, we do not have sufficient information to do the same calculation for P. knowlesi  but with a 24 hour asexual erythrocytic cycle the rate of division will be fast. These periods of 12 or 24 hours seem to be very important to malaria parasites, as you will learn later.

Question 37

Question 11 So, back to our original question… In P. malariae, a single sporozoite can give rise to 15,000 merozoites after 14 days. Do you think this is a rapid rate of multiplication?

Answer 37

The answer is No. This is not really very rapid multiplication in comparison to, say, some bacteria.

Question 38

Erythrocytic stage

Answer 38

The erythrocytic stages have been intensively studied. They are the stages that actually cause the disease. P. falciparum is easy to maintain in culture and therefore the erythrocytic stages are readily obtained.

Question 39

Erythrocytic stage Erythrocytic schizogony

Answer 39

The stages of erythrocytic schizogony are represented as a cycle where arrows lead from one stage to the next. Here, the stages of the cycles are described as a list. Merozoites enter a red blood cell. Merozoites progresses to ring stage. Ring stage progresses to trophozoite. Trophozoite progresses to immature schizont. Immature schizont progresses to mature schizont. Mature schizont causes cell to rupture. Ruptured cell releases merozoites. Merozoites enter a red blood cell

Question 40

Erythrocytic stage

Answer 40

The erythrocytic stages have been intensively studied. They are the stages that actually cause the disease. P. falciparum is easy to maintain in culture and therefore the erythrocytic stages are readily obtained. The merozoites released from blood stage schizonts are very similar to those from exoerythrocytic schizonts.

Question 41

The parasitophorous vacuole

Answer 41

As mentioned earlier, merozoites possess a characteristic apical complex (similar to that seen in the sporozoites), which in this case they use to gain access to the red blood cell. The main organelles of the anterior end of the merozoite are rhoptries, micronemes and dense granules. You will learn more about these later on.

Question 42

The parasitophorous vacuole

Answer 42

The merozoite does not bore through the cell membrane but enters via an invagination of the membrane, which results in the formation of a parasitophorous vacuole. Thus, the parasite resides in a vacuole within the red blood cell.

Question 43

Trophozoites and schizont

Answer 43

Within the parasitophorous vacuole, the merozoite transforms into a feeding stage or trophozoite. On the outside, there is the parasitophorous vacuole membrane, then a very narrow intramembrane space, and then the parasite plasmalemma membrane. Under this in the parasite cytoplasm there are the normal components of a eukaryotic cell including a nucleus containing 14 chromosomes of the genome, a mitochondrion, closely apposed to the apicoplast which is characteristic of the phylum Apicomplexa. Ribosomes populate the cytoplasm.

Question 44

Trophozoites and schizont

Answer 44

The earliest intracellular trophozoites are ring shaped but, as the parasite increases in size, the ring morphology disappears, and the parasite becomes what is sometimes referred to as a mature trophozoite. Nuclear division begins at the trophozoite stage, transforming the trophozoite into a schizont. The mature schizont will rupture and release many merozoites.

Question 45

Trophozoites and schizont Ring, mature trophozoite and schizont stages of P. vivax

Answer 45

The blood cells look pink and are roughly circular in shape.  The slide (or field of view) on the left has one infected cell containing one ring form malaria parasite, stained purple, visible as a thin ring with a thicker patch.  The centre field of view has one large uneven shaped cell in the middle, stained purple, with a differently shaped uneven darker purple shape inside. This is the trophozoite stage of the infection.  The right hand field of view has one schizont, which looks like the red blood cell has lots of tiny purple dots just contained within the cell membrane.

Question 46

Trophozoites and schizont Ring, mature trophozoite and schizont stages of P. falciparum

Answer 46

The left slide (or field of view) is stained peach. The red cells look slightly irregular and five are infected with the ring form, which are visible as darker stained thin rings, with one or two thicker patches. The rings vary from about a quarter to  half the size of the cells.  In one cell, the parasite has divided and two rings can be seen at opposite sides of the cell. The centre field of view is stained purple and at a slightly lower magnification (the red blood cells are smaller), three cells are infected. Two have ring forms stained darker purple, seen as fine rings with two thickened patches about a third the size of the cells.  One has an irregular, almost kite shaped purple stained trophozoite.  The right field of view has blood cells stained green, and is a lower magnification again.  Parasites are stained purple, six cells dotted around the field of view have ring shapes within.  Just below centre is a cell which has just ruptured, this is the schizont.  The cell is nolonger visible, the many parasites (>30) are seen as small purple dots which are just starting to move away from the circle they were enclosed in before the cell ruptured.

Question 47

Trophozoites and schizont

Answer 47

Two things to note: P. vivax mature trophozoites are very irregular in shape (amoeboid) and the two rupturing schizonts of P. falciparum seen in the figure are unusual as they are only rarely present in the peripheral circulation. We shall learn more about that further on.

Question 48

Trophozoites and schizont

Answer 48

The trophozoite begins to feed on haemoglobin, which in the digestive vacuole breaks down into haem and globin components. The parasite cannot digest the haem, which it converts into a haemozoin-like molecule, better known as malaria pigment. The globin is digested and used as a source of amino acids. At the end of the feeding stage, the nucleus divides. This process is repeated a further two to four times, resulting in the production of 8–24 merozoites depending on the species.

Question 49

Bursting of the schizonts

Answer 49

The table you saw earlier in this session showed the number of merozoites produced in the erythrocytic phase per schizont (average) as: 12–18 (16) in P. vivax, 6–10 (8) in P. ovale, 6–10 (8) in P. malariae, 10–24 (16) in P. falciparum, 8–16  (?) in P.knowlesi

Question 50

Bursting of the schizonts

Answer 50

It is usually a synchronous process, so all the schizonts burst at the same time. This bursting releases a number of toxic molecules that are thought to stimulate the body to release factors like TNFα which trigger the periodic fever associated with malaria.

Question 51

Bursting of the schizonts

Answer 51

In P. falciparum infections, the early stages of infection are often asynchronous so that fevers occur with a quotidian (24 hour) cycle rather than tertian (every 48 hours). This may be due in part to there being more than one clone of the parasite which undergo schizogony independently. However, the early fevers do synchronise the infection as the different asexual forms are variously affected by these TNF-activated fevers.

Question 52

Bursting of the schizonts

Answer 52

In  P. vivax , P. ovale, P. malariae and  P. knowlesi infections, all the stages from young trophozoites to mature schizonts are found in the peripheral blood.

Question 53

Bursting of the schizonts

Answer 53

This is not the case for P. falciparum, where apart from the sexual gametocyte stages, generally only red cells infected with young trophozoites are found in the blood (though, as we saw earlier, they can sometimes be seen). Mostly, the older parasites undergoing division export adhesion molecules to the red cell membrane (generally termed protein PfEMP-1) which adhere to endothelial cell surface adhesins like ICAM-1 lining the smaller blood vessels. During the course of the infection, production of these human adhesins is upregulated which exacerbates the effect. This cytoadherence is thus responsible for  sequestration in the deeper circulation and away from the peripheral blood. In the brain, sequestration and related processes are responsible for reducing the blood flow and causing the condition known as cerebral malaria.  There will be more later on how sequestration contributes to malaria pathogenesis.

Question 54

Conclusion

Answer 54

Since the asexual erythrocytic forms of the malaria parasite are responsible for disease, they have been studied in great detail and we know a lot about their structure, biochemistry, molecular biology, genetics, physiology and immunology. In particular, the genomes of several species have been mapped, and the function of some important proteins worked out. All this information is being used to develop vaccines and detect new targets for new drugs, and to elucidate the mechanisms responsible for the pathological processes of disease of malaria. The surface molecules of the merozoite have been particularly well characterised.

Question 55

Conclusion

Answer 55

It is important to note that with each Plasmodium species, not all red blood cells are susceptible to invasion by merozoites, depending on characteristics of the red blood cell membrane proteins, haemoglobin type and to some extent, relative maturity of the red blood cell. These are some of the reasons for the host specificity characteristic of malaria infections. You will learn more about this in Session 3.4 Epidemiology and prevention: Vector control.

Question 56

Question 12 We've mixed up the different stages of the invasion of a red blood cell by a merozoite. Put the stages in the right order (starting with the earliest stages (1) to the latest stages (5)). [choose... | Attachment | Internalisation | Irreversible attachment and junction formation | Orientation | Parasitophorous vacuole formation]

Answer 56

The correct answer is: Attachment Orientation Irreversible attachment and junction formation Parasitophorous vacuole formation Internalisation

Question 57

Question 13 Complete the following sentences by selecting the correct words from the options provided: XXX are two large sacs containing a number of proteins that are discharged through ducts after attachment. XXX are much smaller organelles and are clustered around these ducts. XXX and XXX are involved in attachment and formation of the parasitophorous vacuole. The XX XX are small bodies that release their contents into the parasitophorous vacuole after the merozoite has entered the cell.

Answer 57

The correct answers are: Rhoptries are two large sacs containing a number of proteins that are discharged through ducts after attachment. Micronemes are much smaller organelles and are clustered around these ducts. Rhoptries and micronemes are involved in attachment and formation of the parasitophorous vacuole. The dense granules are small bodies that release their contents into the parasitophorous vacuole after the merozoite has entered the cell.

Question 58

Question 14 What role do rhoptries, micronemes and dense granules play?

Answer 58

All three are membranous structures that are involved in red blood cell invasion. They discharge their contents during the process and change the shape and nature of the invaded cell.

Question 59

Question 15 What is the 'ring stage' of the malaria parasite?

Answer 59

The 'ring stage' is the name given to the first stage after invasion of the red blood cell. It has a rim of cytoplasm containing a conspicuous nucleus and most of its organelles, with very few structures in the centre, hence its appearance.

Question 60

Question 16 How does the early trophozoite (or ring stage) feed?

Answer 60

The trophozoite feeds via a ring-like structure, the cytostome, through which portions of erythrocyte cytoplasm and parasitophorous vacuole are pulled and digested within small vacuoles in the parasite itself.

Question 61

Question 17 What are Maurer's dots (or clefts)?

Answer 61

Maurer's dots (or clefts): form when various membranous structures of the parasite penetrate the erythrocyte cytoplasm and reach the inside of the red cell membrane where the associated proteins are due to be exposed on the red blood cell surface; and are characteristic of P. falciparum-infected red blood cells. Free nuclei are never seen. Killed intracellular parasites have a distinctive appearance when compared with Maurer's dots.

Question 62

Question 18 What is MSP-1, and what is its role?

Answer 62

MSP-1 is the merozoite surface protein-1 (sometimes called merozoite surface antigen-1, MSA-1) thought to be involved in erythrocyte invasion. It is highly conserved across Plasmodium species and is a possible candidate vaccine target.

Question 63

Question 19 It is a simple matter to determine how many divisions are required to produce the average number of merrozoites in asexual blood-stage schizonts of P. malariae, P. vivax and P. falciparum. How do the rates of multiplication compare with the exoerythrocytic stages?

Answer 63

The rate of multiplication in the erythrocytic stages is similar to that seen in the exoerythrocytic phases (once every 24 hours in P.malariae , once every 12 hours in P. vivax , and once every 12 hours in P. falciparum).

Question 64

Question 20 P. knowlesi produces up to 16 merozoites and the rodent malaria parasites such as P. berghei produce about eight.  How many divisions are required to produce these numbers of merozoites?

Answer 64

The rate of multiplication of these parasites is much more rapid than for the other human malarias (division is once every six hours for P. knowlesi and once every eight hours for P. berghei). This is based on the schizogonic cycle in P. knowlesi and P. berghei which is 24 hours. The answer is therefore four.

Question 65

Question 21 What do you think is the importance of these calculations?

Answer 65

The importance of these simple calculations is that they should alert you to the fact that once an infection is established the numbers of parasites in the host increases very rapidly so any delay in treatment could be very serious.

Question 66

Gametocytes

Answer 66

Some merozoites do not mature to schizonts but instead develop into male and female sexual stages or gametocytes. There is now evidence that this involves epigenetic control of gene expression, and small vesicles released from P. falciparum rings and schizonts when their growth is constrained may trigger the development of gametocytes (overview by Del Portillo and Chitnis 2013). The time taken for the gametocytes to mature varies between species and is: 3 to 4 days in P. vivax and P. ovale; 6 to 8 days in P. malariae; and 6 to 12 days in P. falciparum. In P. vivax, P. ovaleand P. malariae, the gametocytes are round. In P. falciparum, they are crescent shaped and are therefore very easy to identify (see the figure below).

Question 67

Gametocytes

Answer 67

The Plasmodium falciparum macrogametocyte is a crescent-shaped structure with a circular darker grainy area in the centre. The Plasmodium ovale macrogametocyte is a grainy, round structure with a darker area on the edge. The Plasmodium malariae macrogametocyte is an grainy oval structure with a darker area at one end. The Plasmodium vivax macrogametocyte is a grainy, round structure with a darker area on the edge.

Question 68

Gametocytes

Answer 68

The long development of P.falciparum gametocytes takes place when they are sequestered out of the peripheral blood mainly in the bone marrow. They pass through five stages that are distinguishable morphologically (shown below) and are released into the circulation at stage 5. While sequestered, the gametocyte-infected red cell membrane is rigid but the stage 5 forms become flexible. If this did not occur, the mature infective gametocytes would not be able to circulate as rigid forms would be removed by the spleen.

Question 69

Gametocytes

Answer 69

Gametocyte development in P. falciparum. Five morphologically different stages are shown here as photographs (top) and schematically (bottom). The earliest stage 1 forms are indistinguishable morphologically from asexual parasites, but from 30 hours after invasion of the red blood cell they are genetically recognisable as committed to sexual development. Stages 1-4 are sequestered mainly in the bone marrow , while stage 5 micro-(male) and macro(female)-gametocytes circulate in the peripheral blood.

Question 70

Gametocytes

Answer 70

First is Stage I, the photograph is of a large pale blue stained circle (red blood cell) with a smaller purple stained slightly uneven shaped and stained circle (parasite). The diagram identifies within the parasite, the nucleus as a smaller circle towards the top, a microtubule as a thin orange rod from the top of the nucleus to the bottom of the parasite, the inner membrane complex as a green line alongside and within a circle beneath the nucleus haemozoin crystals. In stage II the photograph shows the parasite has doubled in size and developed into an almond shape. The purple staining is more intense on one side. The diagram shows the microtubule and membrane complex from point to point of the shape. For stage III the photograph shows half the red blood cell taken up by the parasite, which has changed shape to looks like a flying saucer, the colour of the inside of the red cell is paler. The diagram shows that more microtubules have grown with 4 orange lines along the base of the saucer. The green membrane complex has extended about half way up the sides of the saucer. For stage IV the photograph shows the parasite is again almond shaped with the length about twice as long as the red cell still purple stained there is a slightly pink area towards one of the points (this is the nucleus). The red cell is stretched to accommodate it. The diagram shows that microtubules fill the parasite cell with orange lines from point to point, the inner membrane complex follows inside the parasite cell membrane and all round the cell. The nucleus and haemozoin crystals are in the center of the parasite. 11 small oval shaped osmiophilic bodies are scattered around the inside of the parasite. For stage V the photograph shows two oval shaped pale purple parasites with darker purple lines with pink between. One is slightly curved (like a banana) the curved one is labelled with a female sign, the other with a male sign – these are the gametes. The diagram shows the male (banana shaped one). The ends have become rounded and the microtubules (orange lines) have gone. The inner membrane complex nucleus and osmiophilic bodies remain the same, the haemozoin crystals have doubled in size. The red cell just about remains visible down the concave side.

Question 71

Sporogony

Answer 71

In malaria of man and other mammals, sporogony occurs only in an appropriate mosquito, always a female belonging to the genus Anopheles.

Question 72

Sporogony

Answer 72

When taken up by a susceptible mosquito, the gametocytes escape from the red blood cells within minutes and mature into gametes: female gametes are macrogametes; male gametes are microgametes. The development and release of gametes from gametocytes is called gametogenesis. The development and release of microgametes from microgametocytes is called microgametogenesis or exflagellation.

Question 73

Gametogenesis

Answer 73

During gametogenesis, the macrogamete is released from the macrogametocyte with little change but the microgametocyte undergoes a remarkable transformation.

Question 74

Gametogenesis

Answer 74

The microgametocyte's nucleus divides three times rapidly to produce eight nuclei. Each nucleus becomes associated with a flagellated structure (the microgamete) that breaks free, is very motile, and seeks out and fertilises a macrogamete to form a zygote (also known as an ookinete).

Question 75

Gametogenesis

Answer 75

The fusion of nuclei results in the formation of a diploid nucleus that is quickly restored to the haploid state by a process of meiosis. The ookinete then passes through the peritrophic membrane and midgut epithelium and encysts beneath the basal lamina. It is still not clear what processes are involved in the passage across the midgut and establishment in the basal lamina.

Question 76

Third phase of multiplication

Answer 76

The ookinete, together with host molecules, forms a cyst (the oocyst) and it is here that a third phase of multiplication occurs.

Question 77

Third phase of multiplication

Answer 77

The nucleus of the oocyst divides a number of times resulting in the formation of 4000-10,000 sporozoites. This process of sporogony normally takes from 8-16 days depending on the species and on the temperature; it can sometimes be shorter or longer.

Question 78

Third phase of multiplication

Answer 78

The sporozoites are released from the oocysts into the haemolymph. They arrive at and invade the salivary glands where they remain until the mosquito feeds again. There can be between 10,000 and 200,000 sporozoites in the salivary glands of an infected mosquito but only a small number of these (something between 10 and 100) pass into the saliva and are injected into a new host.

Question 79

Third phase of multiplication

Answer 79

Not all mosquitoes are equally susceptible to infection. Both genetic and environmental factors affect the ability of mosquitoes to support malaria development and transmission, that is, their vector competence (Lefèvre et al. 2013).

Question 80

Question 22 In nature, only a few of the competent mosquitoes that feed on an infected host become infected (less than 5%). How do we define malaria parasite prevalence and parasite intensity in mosquitoes?

Answer 80

Parasite prevalence is the proportion of malaria-exposed mosquitoes with one or more oocysts on the midgut or one or more sporozoites in the salivary glands. Parasite intensity is the number of oocysts on the midgut or the number of sporozoites in the salivary glands.

Question 81

Question 23 Make a list of the main factors that are thought to influence the vector competence of mosquitoes that transmit malaria.

Answer 81

Vector competence – you are asked only to make a list of factors but a few extra comments are included here. Your answer should include all or most of the following:1 Temperature (This has been much studied. In general, the parasite development rate increases as the environmental temperature rises but a peak is reached after which parasite survival drops rapidly.) 2 Humidity 3 Nutrition (For the female mosquito this includes the blood meals it takes and the plants on which it feeds. The breeding sites are also going to be very variable.) 4 Co-infection with gut bacteria, fungi, microsporidia or filarial worms. 5 Mosquito immune responses 6 Age and size of the mosquito. (Old mosquitoes may be more susceptible to infection but do not live long enough to transmit the infection.) 7 Infectiousness of gametocytes ingested with a blood meal. 8 Effect of malaria infection on mosquito reproduction and survival. 9 Mosquito strain (genotypic) differences. 10 Plasmodium strain (genotypic) differences

Question 82

Question 24 What factors are responsible for exflagellation in the mosquito?

Answer 82

A drop in temperature and a rise in pH to more than 7.8 and a ‘mosquito factor’ identified as xanthurenic acid which is a key inducer of exflagellation. You can find out more about xanthurenic acid by reading the paper Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito by Bilker et al.

Question 83

Question 25 You learned in Session 1.3 Parasites and vectors: Plasmodium species and their uses as animal models that it is difficult to infect mosquitoes with P. elongatum. Why might this be so?

Answer 83

Exflagellation of this species seems to be inhibited in Aedes and Anopheles spp.

Question 84

Question 26 When does meiosis occur in the Plasmodium life cycle? a) immediately before fertilisation b) immediately after fertilisation c) during sporozoite formation d) after sporozoite formation

Answer 84

Meiosis occurs immediately after fertilisation in the Plasmodium life-cycle.

Question 85

Question 27 What makes the mammalian malaria life-cycle unusual given the timing of meiosis?

Answer 85

It means that most of the life cycle, in humans as well as in the mosquito, is haploid.

Question 86

Question 28 How does the ookinete penetrate the mosquito midgut?

Answer 86

The short answer is that it is not known for certain how the ookinete penetrates the midgut wall because it appears able to pass both between and through the midgut cells.

Question 87

Question 29 There is a very important difference between sporozoites that are released from an oocyst and those that are expelled from the salivary glands. Complete the statement below by selecting the correct terms from the options below. Sporozoites from XXX cannot infect liver hepatocytes and sporozoites from XXX cannot infect fresh salivary glands.

Answer 87

Sporozoites from an oocyst cannot infect liver hepatocytes and sporozoites from the salivary glands cannot infect fresh salivary glands.

Question 88

Question 30 List the non-human species of Plasmodium that are mentioned in the articles on sporogony.

Answer 88

The most common are P. berghei, P. chabaudi and P. yoelii (rodent malarias) and P. knowlesi, P. elongatum and P. gallinaceum (avian malarias). This shows how important it is to know about animal malaria parasites in order to be able to understand the literature on the subject, and how valuable they have been for research purposes.

Question 89

Question 31 Approximately how long do sporozoites circulate in the blood after injection by a mosquito? a) 5-10 min, b) 30-60 min, c) 60-120 min, d) 3-4h

Answer 89

Sporozoites circulate in the blood for approximately 30-60 minutes after injection by a mosquito

Question 90

Question 32 What is the practical significance of this?

Answer 90

Sporozoites are unlikely to be affected by immune responses or by drugs

Question 91

Question 33 How does a sporozoite enter the liver hepatocyte? Why is it important to know this?

Answer 91

There is evidence that sporozoites gain entry either by penetrating endothelial cells or via Kuppfer cells. Once inside it appears they do not stop in the first hepatocyte they reach but pass through two or three before settling and beginning multiplication. A detailed understanding of this process is important as this helps in design of drugs or vaccines that would block the infection before it could become established.

Question 92

Question 34 Approximately how long does the exoerythrocytic stage last in P. falciparum, P. vivax and P. malariae infections?

Answer 92

P. falciparum 5-7d, P. vivax 6-8d and P. malariae 14-16d

Question 93

Question 35 What is the practical significance of this information?

Answer 93

One reason is that, if antimalarials being taken prophylactically act primarily against the blood stages, this indicates how long after possible exposure to the bites of infected mosquitoes the drug should continue to be taken.

Question 94

Question 36 What mechanisms are involved when a merozoite invades a red blood cell? What is the difference between the initial mechanisms used by P. falciparum and P. vivax ?

Answer 94

The merozoite has to attach to a red blood cell, orient itself so that the apical complex is in contact with the red cell membrane, and induce the formation of a parasitophorous vacuole. P. vivax appears to mainly use the Duffy molecule on the surface of the red blood cell for attachment, whereas P. falciparum other receptors.

Question 95

Question 37 How does a trophozoite obtain the amino acids it needs? What is the consequence of the mechanism used?

Answer 95

Trophozoites feed through a cytostome and ingest host cell haemoglobin. This is broken down into a globin component, which can be used as a source of amino acids, and a haem component, which cannot. The haem is toxic and has to be neutralised; it forms haemozoin or pigment.

Question 96

Question 38 Approximately how long do untreated P. falciparum, P. vivax and P. malariae infections last?

Answer 96

P. falciparum 1-2y, P. vivax 18m-5y and P. malariae 3-50y

Question 97

Question 39 Why is it important to know this?

Answer 97

For a clinician or a patient, it is important to know that a person might still experience bouts of malaria many years after the last exposure to infection or be infectious by donating blood if untreated or inadequately treated. It is important to remember that a person with little or no immunity might die as a result of an infection left untreated, especially P. falciparum.

Question 98

Question 40 What stages of P. falciparum circulate in the peripheral blood? a) Schizonts b) Rings c) Young trophozoites d) Gametocytes

Answer 98

Rings, young trophozoites and gametocytes circulate in the peripheral blood. Schizonts do not circulate in the peripheral blood. Red blood cells containing schizonts of P.falciparum schizonts sequester on the endothelium of blood vessels in major organs of the body. To be accurate, they are occasionally seen in the peripheral blood particularly when the parasitaemia is high.

Question 99

Question 41 Malaria parasite numbers change dramatically during the life-cycle.  What are the challenges, and alternatively the opportunities, for malaria control presented by these number changes?

Answer 99

The massive increase in numbers in the liver and the blood present a big challenge for development of drugs and vaccines. Even if these are say 90% effective, this still leaves a large number of parasites to continue development. On the other hand, where the numbers are small, as occurs at fertilisation and ookinete formation in the mosquito, this is a weak link in the parasites’ life cycle. This is an important reason why vaccines directed at the sexual cycle (the so-called transmission-blocking vaccines) and drugs that will knock-out gametocytes are seen as a priority for development.

Question 100

Summary

Answer 100

Knowledge of the complex life-cycle of Plasmodiumspp. is essential for an understanding of malaria as a disease. It is also essential for devising control measures and for developing vaccines and new drugs. Infection begins when infective sporozoite stages injected by a female Anopheles mosquito enter the bloodstream. The sporozoites enter liver hepatocytes and initiate a phase of multiplication resulting in the formation of an exoerythrocytic schizont producing merozoites that enter the bloodstream and invade red blood cells. Inside the red blood cell, the merozoite becomes a feeding stage (or trophozoite) that digests haemoglobin and multiplies asexually to produce an erythrocytic schizont, which matures to produce a new generation of merozoites. This asexual reproductive cycle is repeated several times. In the human host, some merozoites differentiate into male and female sexual forms called gametocytes. When taken up in the blood meal by a female Anopheles mosquito, the mature gametocytes release extracellular male and female gametes. After fertilisation, a zygote (the ookinete) develops into an oocyst within which sporozoites are formed. Sporozoites enter the salivary glands from which they infect a new host.

Question 101

Glossary Sporozoite

Answer 101

The final product of sporogony (The sexual development of the malaria parasite in a mosquito.). Sporozoites migrate to the salivary gland of the mosquito.

Question 102

Glossary Hepatocytes

Answer 102

A liver cell responsible for synthesis, degradation and storage. Hepatocytes make up 70% of the liver.

Question 103

Glossary Exoerythrocytic schizont

Answer 103

Exoerythrocytic (or pre-erythrocytic) defines the malaria parasite (sporozoite) at the liver stage of infection. Replication within the hepatocytes is often described as exoerythrocytic (or pre-erythrocytic) schizogony. However it is important to note that the term pre-erythrocytic is also used to describe the stages of the malaria parasites (merozoites released from mature hepatic schizonts) that appeared before parasites invade the blood cells.

Question 104

Glossary Merozoites

Answer 104

Part of the asexual reproductive cycle of plasmodia: in malaria, the parasite stage released by mature liver and blood schizonts, and responsible for invading erythrocytes.

Question 105

Glossary Trophozoite

Answer 105

The early erythrocytic feeding stage of the malaria parasite that later becomes a schizont.

Question 106

Glossary Erythrocytic schizogony

Answer 106

The phase of asexual multiplication of the malaria parasite in hepatocytes or erythrocytes. The final product is a schizont which on maturation ruptures to release merozoites.

Question 107

Glossary Gametocytes

Answer 107

Immature male (microgamete) or female (macrogamete) ) cells which develop into reproductive gametes. In the malaria parasite, these stages occur in the blood of the vertebrate host and are taken up by a mosquito.

Question 108

Glossary Zygote

Answer 108

The immediate product of fertilisation, also called the ookinete.