EXPERIMENTAL
PARASITOLOGY
Plasmodium
74, l-10 (1992)
berghei: In Vivo Generation Mutants and Non-Gametocyte
and Selection of Karyotype Producer Mutants
C. J. JANSE, J. RAMESAR, F. M. VAN DEN BERG,* AND B. MONS Laboratory
of Parasitology, *Department
University of Leiden, P.O. Box 9605, 2300 RC Leiden, The Netherlands; of Pathology, AMC, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
and
JANSE, C. J., RAMESAR, J., VAN DEN BERG, F. M., AND MONS, B. 1992. Plasmodium bergheic In vivo generation and selection of karyotype mutants and non-gametocyte producer mutants. Experimental Parasitology 74, l-10. We previously reported that karyotype and gametocyte-producer mutants spontaneously arose during in viva asexual multiplication of Plasmodium berghei. Here we studied the rate of selection of these mutants in viva. Gametocyte production and karyotype pattern were established at regular intervals during prolonged periods of asexual multiplication of clone 8417 of P. berghei. We found
that karyotype mutants and mutants which do not produce gametocytes can replace the original high-producer parasites of clone 8417 within several weeks. The time at which mutants became predominant in the population in different experiments, however, differed greatly. Mutants with intermediate or low gametocyte production were not found. In experimentally mixed infections, containing parasites from two clones from different strains (clone 8417 of the ANKA strain; clone 1 of the K173 strain), high-producer parasites of clone 8417 were overgrown by parasites of the nonproducer clone. Nonproducer mutants from the originally high-producer clone 8417, however, were able to coexist with parasites of the nonproducer clone. These results demonstrate that in our experiments nonproducer parasites had a strong selective advantage during asexual multiplication compared to high producers. All karyotype mutants which became predominant in our experiments were nonproducers. In two experiments a change in karyotype coincided with the loss of gametocyte production which may suggest a causal relationship between these events. 8 1~2 Academic Press, Inc.
Plasmodium bergheic Asexual multiplication; INDEX DESCRIPTORS AND ABBREVIATIONS: Gametocyte production; Nonproducer mutants; Karyotype mutants; Selection; Field inversion gel electrophoresis (FIGE); Kilobase (kb).
ation of genetic diversity in P. falciparum (Walliker 1989; Kemp et al. 1990). Increasing our knowledge of the molecular mechanisms involved in genetic variation would help us to understand the parasite’s capacity to adapt to changing environments and to circumvent control measurements. We use the rodent parasite P. berghei as a model to study the generation of genetic diversity during asexual (mitotic) multiplication in vivo. We previously reported that mutants spontaneously arose during asexual multiplication, showing altered karyotypes and gametocyte production (Janse et al. 1989b). We found evidence that recombination between chromosomes during mi-
Extensive genetic variation is found between isolates of the human malaria parasite Plasmodium falciparum (Creasey et al. 1990; Kemp et al. 1990). Also within one isolate genetic differences exist between parasites. Genetic diversity has been identified for antigens, proteins, enzymes, size of chromosomes, susceptibility to drugs (for review see Kemp et al. 1990), and capacity to produce gametocytes (Bhasin and Trager 1984; Burkot et al. 1984; Graves et al. 1984; Janse et al. 1989b). Mutation and genetic recombination during meiosis appear to play an important role in the gener1
OOl4-4894/92$3.00 Copyright Q 1992 by Acndemic Press. Inc. All rights of reproduction in any form reserved.
2
JANSE
tosis may play a role in generation of chromosome size polymorphism (Ponzi et al. 1990; Pace et al. 1990). This suggests that recombination during mitosis may also provide a means for the generation of genetic diversity in malaria parasites. In spite of frequent production of karyotype mutants, populations of asexually propagating parasites generally appeared to be homogeneous with regard to the karyotype of the parasites (Janse et al. 1989a). This suggests that particular mutants have a selective growth advantage, which enables these mutants to predominate. To get more information about the process of selection of mutants we studied relative changes in the proportion of karyotype mutants and nongametocyte producer mutants during prolonged periods of asexual multiplication of a high-producer clone. In addition, we established mixed infections in mice of two clones, which differ in their karyotype and gametocyte production and quantitated the proportion of each clone in the mixed populations during prolonged periods of multiplication in mice. MATERIALS
AND METHODS
Parasites. We used clone 8417 of the ANKA strain and clone 1 of the K173 strain of P. berghei. These clones and strains are described by Janse et al. (1989a,b). Asexual multiplication of a high gametocyte producer clone. In eight independent experiments the high producer clone 8417 was maintained in mice for a period of 25 to 65 weeks. In each experiment parasites were weekly mechanically transmitted to noninfected mice by intraperitoneal injection of lo6 infected erythrocytes. At regular intervals gametocyte production and the karyotype pattern of the population were established (see below). The experiment was terminated when gametocyte production of the population was undetectable for a period of 4 weeks. Mixed infections of a high gametocyte producer clone (8417) and a nonproducer clone (I). We established six mixed infections by infecting six mice simultaneously with 0.5 x lo6 parasites of clone 8417 and 0.5 x lo6 parasites of clone 1. These mixed infections were maintained in Swiss mice for a period of 25 weeks by mechanical transmission. Each week gametocyte production and the karyotype pattern of the
ET AL.
mixed infections were established. Infectivity to mosquitoes was tested at regular intervals by feeding susceptible Anopheles atroparvus mosquitoes on the mice carrying a mixed infection. Mosquitoes were fed on Day 4 or 5 after infection and 20 days later they were allowed to feed on a noninfected mouse to test if the mosquitoes had been infected by parasites of clone 8417. Gametocyte production. Gametocyte production is defined as the percentage of parasites which developed into gametocytes in synchronized infections in mice under standardized conditions as described by Mons et al. (1985). Four classes were distinguished: 15-25% (3); 5-15% (2), O-5% (1); and 0% (0). Karyotype pattern. The karyotype pattern of the populations was established by Field inversion gel electrophoresis (FIGE). Infected blood was obtained by heart puncture 7 days after infection of the mouse. Blood cells were lysed in 155 mM NH&l, 10 mM KHCO,, and 1 mM EDTA for 20 min at 4°C. The parasites were pelleted for 10 min at 350g and suspended in 5 ml TNE (50 mM Tris, 5 mM EDTA, 100 ELM NaCl). To remove a large proportion of contaminating leucocytes, this suspension was layered on top of a 48% (v/v) mixture of Nycodenz and PBS and centrifuged at 200 g for 30 min. The parasites from the interface were collected, washed with PBS, and pelleted. The pellet was added to an equal volume of molten 2% low gel temperature agarose (Bio-Rad) at 37°C and was cast into 1 x 5 x S-mm blocks which were treated with proteinase K as described (Janse et al. 1989b). We used FIGE, applying a Chromopulse TM switching unit as described by Janse er al. (1989b). Electrophoresis conditions are described in the legends to the figures. FIGE gels were stained with ethidium bromide, photographed, and Southern blotted onto nitrocellulose membranes by previously described methods (Maniatis et al. 1982). Blots were hybridized under standard conditions at 60°C with the 2.3 kilobase (kb) subtelomeric repeat probe, a telomeric probe, or with chromosome-specific probes described by Ponzi et al. (1990). These probes were labeled by random priming. RESULTS
Generation and selection of nonproducer mutants in a high gametocyte producer clone. We mechanically passaged the high-
producer clone 8417 in mice in eight independent experiments for prolonged periods, up to more than a year. In all experiments mutants that did not produce gametocytes (nonproducer mutants) arose and replaced the gametocyte producer par-
P. berghei: IN VIVO GENERATION
asites. This eventually resulted in the complete loss of gametocyte production from the population. In three experiments gametocyte production during the period of asexual multiplication was established at irregular intervals. Therefore, the results of these experiments which were published previously (Janse et al. 1989b) gave no information about the exact time and rate of the loss of gametocyte production. In the other five experiments we established gametocyte production at 2- to 5-week intervals. The results of these experiments show that a cloned population can change within several weeks from a high gametocyte production (high-producer clone) into a population without gametocytes (Fig. 1). The time at which gametocyte production decreased in the different experiments differed to a great extent. Gametocyte production can be lost after 15-20 weeks or can remain stable for almost a year (Fig. 1). In two experiments we cloned parasites from the populations by the method of limiting dilution during the period in which gametocyte production was decreasing. From 15 of these clones we established the gametocyte production. Three clones showed a
period
of asexual
multiplication
(weeks)
FIG. 1. The loss of gametocyte production during asexual multiplication in vivo of a high-producer clone (8417) of Plasmodium berghei in five independent experiments. Gametocyte production was established under standardized conditions in synchronized infections in mice and defined as the percentage of parasites developing into mature gametocytes. Four classes were distinguished: 0, 0%; 1, O-5%; 2, 5-15%; 3, 1525%.
AND
SELECTION
OF MUTANTS
3
high production of gametocytes (H-25%), comparable to that of the original clone 8417, while the others were nonproducers. We never found clones with an intermediate gametocyte production (class 1 or 2). In a previous paper, however, we reported clones with an intermediate gametocyte production of l-5% (Janse et al. 1989b). In these experiments the presence of gametocytes had been established by counting gametocytes in Giemsa-stained slides. Since in P. berghei old trophozoites can be wrongly identified as (young) gametocytes (Mons et al. 1987), we decided to reexamine gametocyte production of these clones. In three synchronized infections of each clone we did not find fully mature gametocytes in Giemsa-stained slides nor could we detect exflaggelation after stimulation of gametogenesis by described methods (Janse et al. 1985), indicating that these clones are nonproducers in contrast to our earlier report. Generation and selection of karyotype mutants in a high-producer clone. In four
out of eight experiments karyotype mutants replaced the parasites with the original karyotype of clone 8417. Karyotypes of the mutants were different from each other (Fig. 2) and all mutants were nongametocyte producers. In two experiments we were able to follow the overgrowing of the original parasites by the karyotype mutants. Within several weeks the population changed from the original karyotype into the mutant karyotype as shown in Fig. 3. The increase of parasites with the mutant karyotype coincided with the decrease of gametocyte production, suggesting that the loss of gametocyte production and a change in the karyotype occurred simultaneously in the same parasite. Competition between parasites in experimentally mixed infections. We established
six independent mixed infections of a high gametocyte-producer clone (8417) and a nonproducer clone (1) and quantitated rel-
JANSE
1234
1
2
ET AL.
3
4
5 3.5 Mb
0.6 2. Karyotype of four mutants (1-I) which replaced the parent parasites of clone 8417 (5) of Plasmodium berghei in four independent experiments. Major changes of the karyotype are indicated by arrows. FIGE was performed at 3.5 V/cm with linearly increasing forward pulse from 40-350 set and a forward/backward ratio of 3/l. FIG.
ative changes in the proportion of each clone in the mixed populations during 25 weeks of asexual multiplication. Recently we reported detailed comparisons of several growth characteristics of these clones (Janse et al. 1989b). The duration of the schizogonic cycle, reticulocyte preference, and multiplication in viva did not significantly differ between these parasites. The clearly different karyotype allows easy discrimination between these clones in mixed infections (Fig. 4). Chromosome 7 of clone 8417 shows strong hybridization with the 2.3-kb repeat probe (more than 100 repeats; Ponzi et al. 1990), while chromosome 7 of clone 1 does not contain 2.3-kb repeats and is 0.5 megabases smaller. As a result we
were able to detect only 1% parasites of clone 8417 in a serial dilution of these parasites with parasites of clone 1, using FIGE karyotypes hybridized with the 2.3-kb probe (Fig. 5, lanes 3-7). Figure 4 shows the karyotype pattern of the six mixed infections after 1 week of multiplication. The karyotypes of both clones are visible and from the staining intensity of the individual chromosomes it appears that both clones were present in a ratio of 1: 1. In five out of six mixed infections high-producer parasites of clone 8417 were overgrown by nonproducer parasites of clone 1 within 9 to I5 weeks, as was shown by the disappearance of the karyotype of clone 8417. See for example the dis-
P. berghei:
IN VIVO
Al A2
GENERATION
Al
AND
SELECTION
92 91
A2
OF MUTANTS
5
92 91
5 FIG. 3. Karyotype patterns of two populations of clone 8417 of Plasmodium berghei just before (Al, Bl) and just after (A2, B2) the loss of gametocyte production. Hybridization with chromosomespecific probes shows that in population A nonproducer mutants with a smaller chromosome 5 replaced the high-producer parasites of clone 8417. In population B nonproducer mutantswith a smaller chromosome 7 became predominant. Al, Week 48; A2, Week 53; Bl, Week 12; B2, Week 16.
appearance of hybridization of the 2.3-kb probe with chromosome 7 of clone 8417 in two experiments in Fig. 5 (lanes 8-15). The last week in which we could detect parasites of clone 8417 is shown in Table I. In all experiments there was no abrupt change in the proportion of the two clones but the proportion of clone 8417 decreased gradually. As expected, the disappearance of parasites of clone 8417 coincided with loss of gametocyte production and infectivity to mosquitoes (Table I). Mosquitoes, however, still became infected after feeding on mixed infections containing less than 1% parasites of the high-producer clone. In one mixed infection, parasites of the high-producer clone remained present during the period of 25 weeks as was shown by the karyotype pattern of the population
(Fig. 6). Surprisingly, gametocyte production and infectivity to mosquitoes were completely lost after Week 18 in spite of the presence of parasites of clone 8417. This suggests that in this particular infection parasites of clone 8417 had lost the capacity to produce gametocytes and were able to coexist with nonproducer parasites of clone 1. To test this hypothesis we cloned parasites by the method of limiting dilution from the population at Week 25. Out of a total of 25 clones, 7 exhibited the same karyotype as clone 8417 (except for a small change in the size of chromosome 7). All these clones were nonproducers. We followed this mixed infection for another period of 15 weeks in four independent experiments. In two of these experiments nonproducer parasites of clone 8417 predominated the non-
JANSE ET AL.
12345678
8417 ::@I
35Mb
06 4. Karyotype patterns of six mixed infections (lanes 2-7) of clone 8417 (ANKA strain) and clone 1 (K173 strain) of Plasmodium berghei after 1 week of asexual multiplication in mice. Lanes 1 and 8 show the karyotype pattern of experimentally mixed parasites of both clones in a ratio of 1:l). Arrows show the position of chromosome 7 in both clones. FIGE was performed at 3.5 V/cm, with linearly increasing forward pulse from 60-500 set and a forward/backward ratio of 311. Schematic representation of the karyotype of clone 1 and clone 8417 as established by Ponzi et al. (1990). FIG.
1990). The latter process may provide a means for the generation of genetic diversity in addition to gene reassortment and recombination between homologous chromosomes during sexual development of P. DISCUSSION fulciparum (Corcoran et al. 1988; Sinnis In P. berghei karyotype mutants and and Wellems 1988; Vernick et al. 1988). Parasites can readily lose the capacity to nongametocyte producer mutants spontaneously arise and rapidly predominate dur- produce gametocytes during asexual multiing asexual multiplication in vivo (this pa- plication in vivo. We showed here that the per, Janse et al. 1989b). Recently we found decrease of gametocyte production is the not only that karyotype mutants arise as a result of competition between high- and result of deletion of DNA sequences, as nonproducer mutants. Nonproducer mutants rapidly predominated over highwas shown to occur in vitro in P. fulciparum (Corcoran et al. 1988), but also that producer parasites of the parent clone. It recombinational events occur during mito- seems logical that the production of gamesis, resulting in the enlargement of certain tocytes by asexually dividing parasites is a chromosomes (Ponzi et al. 1990; Pace et al. selective disadvantage in terms of overall producers of clone 1, while in the other two experiments parasites of clone 1 became predominant.
P. ber&ei:
12
IN VIVO GENERATION
3 4
5
6
7
7
AND SELECTION OF MUTANTS
8
9 10 11 12 13 14
15
FIG. 5. Karyotype pattern and hybridization pattern with the 2.3-kb probe of mixed infections of clone 8417 and clone 1 of Plasmodium berghei. Arrows show the position of chromosome 7 of both clones. Lane 1, clone 8417; lane 2, clone 1; lanes 3-7, experimentally mixed parasites of clone 8417 and clone 1 in the ratio of 1:l (lane 3), 1:9 (lane 4), 199 (lanes 5,6), and 1:999 (lane 7). Lanes 8-12, mixed infection number 4 at Weeks 4, 8, 10, 12, and 14, respectively. Lanes 13-15: mixed infection number 5 at Weeks 4, 6, and 14, respectively. FIGE was performed at 3.5 V/cm, with linearly increasing forward pulse from 40-350 set and a forward/backward ratio of 3/l.
multiplication rate, since merozoites which develop into gametocytes produce no progeny for the next asexual cycle. We cannot conclude that the difference in gametocyte production is the only factor influencing the competition between the mutants, although it was the only difference detected.
Clones with intermediate gametocyte production were not found in our experiments, suggesting a simple genetic mechanism by which gametocyte production was clones showing a lost. In P. fulciparum, range of different capacities to produce gametocytes have been isolated (Burkot et al.
JANSE
TABLE 1 Disappearance of Gametocyte Production in a Clone of Plasmodium berghei (8417) with a High Capacity of Gametocyte Production Mixed with a Nongametocyte Producing Clone (1) Clone 8417 undetectable from Week” 10 13 13 15 9 clone 8417 remained present
Mosquitoes infected with clone 8417 in Week
Mosquitoes not infected from Week
5, 11 8, 11, 13, 14, 17 8 4, 5, 12, 14 11, 14
14 (4 exp.) 18 (5 exp.) 20 (3 exp.) 20 (4 exp.) 16 (4 exp.)
12, 14
18 (5 exp.)
Nofe. Six mixed infections of these clones were followed over a period of 25 weeks. a This has been established on the basis of the hybridization signal of chromosome 7 of done 8417 with the 2.3-kb probe (see Fig. 5).
1984; Graves et al. 1984) and Said et al. (1986) found P. berghei clones with a very low gametocyte production. In these studies, however, clones were isolated directly from mixed populations rather than being allowed to spontaneously appear as in our 1 2
ET AL.
experiments. It remains to be investigated whether or not intermediate clones exist in our mixed populations. We reported earlier that no clear correlation exists between the karyotype pattern and the capacity to produce gametocytes (Janse et al. 1989b). Although four karyotype mutants described in this paper were nonproducers, suggesting a relationship, we also found nonproducer mutants with no detectable alterations in the karyotype. On the other hand, in two experiments described here the loss of gametocyte production and a change in the karyotype (deletions in chromosomes 7 or 5) occurred simultaneously in the same parasite, which may suggest a causal relationship between the two events. In our experiments, karyotype mutants which do not produce gametocytes are more likely to become dominant in the population in comparison with other karyotype mutants, since nonproducers have a strong selective advantage. Therefore, karyotype mutants with a high gametocyte production will not easily be detected. By cloning parasites from a population of the high-producer clone 8417, we
3456789
FIG. 6. Karyotype pattern and hybridization pattern with the 2.3-kb probe of mixed infection number 6 (clone 8417 and clone 1 of Phmodium berg&i) at Weeks 1, 5, 10, 15, 20, and 25, respectively (lanes l-7). Lane 8, clone 8417; lane 9, clone 1. Arrows show the position of chromosome 7 of both clones.
9
P. berghei: IN VIVO GENERATION AND SELECTION OF MUTANTS
have found karyotype mutants with a high gametocyte production (results not shown), indicating that such mutants do arise but do not become dominant in the population. In nature, the transmission of malaria parasites by their vector mosquitoes is essential for the survival of the parasite. Therefore, one would expect genetic factors controlling the production of gametocytes in Plasmodium to be stable. In P. berghei the gene or set of genes which are responsible for the switch to sexual differentiation are in fact not stable at all. This instability may be explained by the laboratory conditions under which the parasites are maintained in our experiments. However, transmission of malaria parasites in nature is not just a matter of producing as many gametocytes as possible, but is a complex interplay between the parasite and its different hosts. Therefore, selection of a stable and high gametocyte production during prolonged periods of asexual multiplication in the vertebrate host may be absent in nature. Mosquito transmission is a strong selective mechanism, so that only gametocyte-producer parasites will be transmitted to subsequent hosts. Thus, every sporozoite-induced infection will start with an optimal gametocyte production. The process of the loss of gametocyte production we described here resembles the loss of the capacity to cytoadhere in P. fulciparum parasites cultured in vitro. The capacity to cytoadhere is an essential feature in the vertebrate host but can be lost during mitotic multiplication in vitro. Interestingly, it has been found that cytoadherence-negative mutants, which arise during asexual multiplication, were nongametocyte producers and showed a deletion on chromosome 9 (Forsyth et al. 1990; K. P. Forsyth, personal communication). In the light of the observations described here it would be very interesting to know if there is a causal relationship between this change of the karyotype, the loss of cytoadherence, and the loss of gametocyte production.
ACKNOWLEDGMENT This work was carried out under EEC contract TS20126-l(NL) in the framework of the program “Science and Technology for Development.” REFERENCES V. K., AND TRACER, W. 1984. Gametocyte forming and nongametocyte forming clones of P/as-
BHASIN,
modium falciparum. American Journal Medicine and Hygiene 3, 534537. BURKOT,
T. R., WILLIAMS,
J. L.,
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AND SCHEIDER,
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1984. Infectivity to mosquitoes of Plasmodium falciparum clones grown in vitro from the same isolate. Transactions of the Royal Society for Tropical Medicine and Hygiene 18, 339-341. CORCORAN, L. M., THOMPSON, J. K., WALLIKER, D., AND KEMP, D. J. 1988. Homologous recombination
within subtelomeric repeat sequences generates chromosome size polymorphisms in Plasmodium falciparum. Cell 53, 807-813. CREASY, A., FENTON, B., WALKER, A., THAITONG, S., OLIVEIRA,
S., MUTAMBU,
S., AND WALLIKER,
D. 1990. Genetic diversity of Plasmodium falciparum shows geographical variation. American Journal of Tropical Medicine
and Hygiene 42, 403-
413. FORSYTH, K. P., KARAMALIS, F., BIGGS, B., WYCHERLEY, K., CULVENOR, J., BROWN, G. V., WILKINSON, D., BOYD, A. A., AND KEMP, D. J.
1990. Characterisation of the cytoadherence phenotype of cloned lines of Plasmodium falciparum. VII. International Congress of Parasitologie. Bulletin de la Societe Francaise de Parasitologie (Suppl. l), 102. GRAVES, P. M., CARTER, R., AND MCNEILL, K. M. 1984. Gametocyte production in cloned lines of Plasmodium falciparum. American Journal of Tropical Medicine and Hygiene 33, 1045-1050. JANSE, C. J., BOORSMA, E. G., GROBBEE, M. J., AND MONS, B.
RAMESAR,
J.,
1989a. Host cell
specificity and schizogony of Plasmodium berghei under different in vitro conditions. International Journal for Parasitology 19, 509-5 14. JANSE, C. J., BOORSMA, E. G., RAMESAR, J., VAN VIANEN, PH., VAN DER MEER, R., ZENOBI, P., CASAGLIA, O., MONS, B., AND VAN DEN BERG, F. M. 1989b. Plasmodium berghei: Gametocyte
production, DNA content, and chromosome-size polymorphisms during asexual multiplication in vivo. Experimental
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JANSE, C. J., ROUWENHORST, R. J., KLOOSTER, P. F. J., VAN DER KAAY, H. J., AND OVERDULVE,
J. P. (1985). Development of Plasmodium berghei ookinetes in the midgut of Anopheles atroparvus mosquitos and in vitro. Parasitology 91, 219-225. KEMP.
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A. F..
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1990. Genetic diversity in Plasmodium falciparum. in Parasitology 29, 75-149. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. 1982. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. MONS, B., JANSE, C. J., BOORSMA, E. G., AND VAN H. J. 1985. Synchronized erythrocytic DER KAAY, schizogony and gametocytogenesis of Plasmodium berghei in-vivo and in-vitro. Parasitology 91, 423Advances
430. PACE, T., PONZI, M., B., AND FRONTALI,
DORE, E., JANSE, C. J., MONS,
C. 1990. Long insertions within telomeres contribute to chromosome-size polymorphism in Plasmodium berghei. Molecular and Cellular Biology, 12, 6759-6764.
PONZI, M., JANSE, C. J., DORE, E., SCOTTI, R., PACE, T., RETERINK, T. J. F., VAN DER BERG, F. M., AND MONS, B. 1990. Generation of chromosome-size
polymorphism during in vivo mitotic multiplication of P. berghei involves both loss and addition of subtelomeric repeat sequences. Molecular and Biochemical Parasitology 41, 73-82.
A., TIMPERMAN, G., AND WERY, M. 1986. Influence de la maintenance d’une souche de Plasmodium berghei ANKA sur la gametocytgenese. Annales Society beige de Medicine tropicale 66, 123131. SINNIS, P., AND WELLEMS, T. E. 1988. Long-range restriction maps of Plasmodium falciparum chromosomes: Crossing over and size variation among geographically distant isolates. Genomics 3, 287-295. VANDERBERG, J. P., AND GWADZ, R. W. 1980. The transmission by mosquitoes of plasmodia in the laboratory. In “Malaria” (J. P. Kreier, Ed.), Vol. 2, pp. U&234. Academic Press, New York. VERNICK, K. D., WALLIKER, D., AND MCCUTCHAN, T. F. 1988. Genetic hypervariability of telomererelated sequences is associated with meiosis in PlasSAID,
modium falciparum. 6973-6985.
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Received 27 November 1990; accepted with revision 20 July 1991
PARASITOLOGY
Plasmodium
74, l-10 (1992)
berghei: In Vivo Generation Mutants and Non-Gametocyte
and Selection of Karyotype Producer Mutants
C. J. JANSE, J. RAMESAR, F. M. VAN DEN BERG,* AND B. MONS Laboratory
of Parasitology, *Department
University of Leiden, P.O. Box 9605, 2300 RC Leiden, The Netherlands; of Pathology, AMC, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
and
JANSE, C. J., RAMESAR, J., VAN DEN BERG, F. M., AND MONS, B. 1992. Plasmodium bergheic In vivo generation and selection of karyotype mutants and non-gametocyte producer mutants. Experimental Parasitology 74, l-10. We previously reported that karyotype and gametocyte-producer mutants spontaneously arose during in viva asexual multiplication of Plasmodium berghei. Here we studied the rate of selection of these mutants in viva. Gametocyte production and karyotype pattern were established at regular intervals during prolonged periods of asexual multiplication of clone 8417 of P. berghei. We found
that karyotype mutants and mutants which do not produce gametocytes can replace the original high-producer parasites of clone 8417 within several weeks. The time at which mutants became predominant in the population in different experiments, however, differed greatly. Mutants with intermediate or low gametocyte production were not found. In experimentally mixed infections, containing parasites from two clones from different strains (clone 8417 of the ANKA strain; clone 1 of the K173 strain), high-producer parasites of clone 8417 were overgrown by parasites of the nonproducer clone. Nonproducer mutants from the originally high-producer clone 8417, however, were able to coexist with parasites of the nonproducer clone. These results demonstrate that in our experiments nonproducer parasites had a strong selective advantage during asexual multiplication compared to high producers. All karyotype mutants which became predominant in our experiments were nonproducers. In two experiments a change in karyotype coincided with the loss of gametocyte production which may suggest a causal relationship between these events. 8 1~2 Academic Press, Inc.
Plasmodium bergheic Asexual multiplication; INDEX DESCRIPTORS AND ABBREVIATIONS: Gametocyte production; Nonproducer mutants; Karyotype mutants; Selection; Field inversion gel electrophoresis (FIGE); Kilobase (kb).
ation of genetic diversity in P. falciparum (Walliker 1989; Kemp et al. 1990). Increasing our knowledge of the molecular mechanisms involved in genetic variation would help us to understand the parasite’s capacity to adapt to changing environments and to circumvent control measurements. We use the rodent parasite P. berghei as a model to study the generation of genetic diversity during asexual (mitotic) multiplication in vivo. We previously reported that mutants spontaneously arose during asexual multiplication, showing altered karyotypes and gametocyte production (Janse et al. 1989b). We found evidence that recombination between chromosomes during mi-
Extensive genetic variation is found between isolates of the human malaria parasite Plasmodium falciparum (Creasey et al. 1990; Kemp et al. 1990). Also within one isolate genetic differences exist between parasites. Genetic diversity has been identified for antigens, proteins, enzymes, size of chromosomes, susceptibility to drugs (for review see Kemp et al. 1990), and capacity to produce gametocytes (Bhasin and Trager 1984; Burkot et al. 1984; Graves et al. 1984; Janse et al. 1989b). Mutation and genetic recombination during meiosis appear to play an important role in the gener1
OOl4-4894/92$3.00 Copyright Q 1992 by Acndemic Press. Inc. All rights of reproduction in any form reserved.
2
JANSE
tosis may play a role in generation of chromosome size polymorphism (Ponzi et al. 1990; Pace et al. 1990). This suggests that recombination during mitosis may also provide a means for the generation of genetic diversity in malaria parasites. In spite of frequent production of karyotype mutants, populations of asexually propagating parasites generally appeared to be homogeneous with regard to the karyotype of the parasites (Janse et al. 1989a). This suggests that particular mutants have a selective growth advantage, which enables these mutants to predominate. To get more information about the process of selection of mutants we studied relative changes in the proportion of karyotype mutants and nongametocyte producer mutants during prolonged periods of asexual multiplication of a high-producer clone. In addition, we established mixed infections in mice of two clones, which differ in their karyotype and gametocyte production and quantitated the proportion of each clone in the mixed populations during prolonged periods of multiplication in mice. MATERIALS
AND METHODS
Parasites. We used clone 8417 of the ANKA strain and clone 1 of the K173 strain of P. berghei. These clones and strains are described by Janse et al. (1989a,b). Asexual multiplication of a high gametocyte producer clone. In eight independent experiments the high producer clone 8417 was maintained in mice for a period of 25 to 65 weeks. In each experiment parasites were weekly mechanically transmitted to noninfected mice by intraperitoneal injection of lo6 infected erythrocytes. At regular intervals gametocyte production and the karyotype pattern of the population were established (see below). The experiment was terminated when gametocyte production of the population was undetectable for a period of 4 weeks. Mixed infections of a high gametocyte producer clone (8417) and a nonproducer clone (I). We established six mixed infections by infecting six mice simultaneously with 0.5 x lo6 parasites of clone 8417 and 0.5 x lo6 parasites of clone 1. These mixed infections were maintained in Swiss mice for a period of 25 weeks by mechanical transmission. Each week gametocyte production and the karyotype pattern of the
ET AL.
mixed infections were established. Infectivity to mosquitoes was tested at regular intervals by feeding susceptible Anopheles atroparvus mosquitoes on the mice carrying a mixed infection. Mosquitoes were fed on Day 4 or 5 after infection and 20 days later they were allowed to feed on a noninfected mouse to test if the mosquitoes had been infected by parasites of clone 8417. Gametocyte production. Gametocyte production is defined as the percentage of parasites which developed into gametocytes in synchronized infections in mice under standardized conditions as described by Mons et al. (1985). Four classes were distinguished: 15-25% (3); 5-15% (2), O-5% (1); and 0% (0). Karyotype pattern. The karyotype pattern of the populations was established by Field inversion gel electrophoresis (FIGE). Infected blood was obtained by heart puncture 7 days after infection of the mouse. Blood cells were lysed in 155 mM NH&l, 10 mM KHCO,, and 1 mM EDTA for 20 min at 4°C. The parasites were pelleted for 10 min at 350g and suspended in 5 ml TNE (50 mM Tris, 5 mM EDTA, 100 ELM NaCl). To remove a large proportion of contaminating leucocytes, this suspension was layered on top of a 48% (v/v) mixture of Nycodenz and PBS and centrifuged at 200 g for 30 min. The parasites from the interface were collected, washed with PBS, and pelleted. The pellet was added to an equal volume of molten 2% low gel temperature agarose (Bio-Rad) at 37°C and was cast into 1 x 5 x S-mm blocks which were treated with proteinase K as described (Janse et al. 1989b). We used FIGE, applying a Chromopulse TM switching unit as described by Janse er al. (1989b). Electrophoresis conditions are described in the legends to the figures. FIGE gels were stained with ethidium bromide, photographed, and Southern blotted onto nitrocellulose membranes by previously described methods (Maniatis et al. 1982). Blots were hybridized under standard conditions at 60°C with the 2.3 kilobase (kb) subtelomeric repeat probe, a telomeric probe, or with chromosome-specific probes described by Ponzi et al. (1990). These probes were labeled by random priming. RESULTS
Generation and selection of nonproducer mutants in a high gametocyte producer clone. We mechanically passaged the high-
producer clone 8417 in mice in eight independent experiments for prolonged periods, up to more than a year. In all experiments mutants that did not produce gametocytes (nonproducer mutants) arose and replaced the gametocyte producer par-
P. berghei: IN VIVO GENERATION
asites. This eventually resulted in the complete loss of gametocyte production from the population. In three experiments gametocyte production during the period of asexual multiplication was established at irregular intervals. Therefore, the results of these experiments which were published previously (Janse et al. 1989b) gave no information about the exact time and rate of the loss of gametocyte production. In the other five experiments we established gametocyte production at 2- to 5-week intervals. The results of these experiments show that a cloned population can change within several weeks from a high gametocyte production (high-producer clone) into a population without gametocytes (Fig. 1). The time at which gametocyte production decreased in the different experiments differed to a great extent. Gametocyte production can be lost after 15-20 weeks or can remain stable for almost a year (Fig. 1). In two experiments we cloned parasites from the populations by the method of limiting dilution during the period in which gametocyte production was decreasing. From 15 of these clones we established the gametocyte production. Three clones showed a
period
of asexual
multiplication
(weeks)
FIG. 1. The loss of gametocyte production during asexual multiplication in vivo of a high-producer clone (8417) of Plasmodium berghei in five independent experiments. Gametocyte production was established under standardized conditions in synchronized infections in mice and defined as the percentage of parasites developing into mature gametocytes. Four classes were distinguished: 0, 0%; 1, O-5%; 2, 5-15%; 3, 1525%.
AND
SELECTION
OF MUTANTS
3
high production of gametocytes (H-25%), comparable to that of the original clone 8417, while the others were nonproducers. We never found clones with an intermediate gametocyte production (class 1 or 2). In a previous paper, however, we reported clones with an intermediate gametocyte production of l-5% (Janse et al. 1989b). In these experiments the presence of gametocytes had been established by counting gametocytes in Giemsa-stained slides. Since in P. berghei old trophozoites can be wrongly identified as (young) gametocytes (Mons et al. 1987), we decided to reexamine gametocyte production of these clones. In three synchronized infections of each clone we did not find fully mature gametocytes in Giemsa-stained slides nor could we detect exflaggelation after stimulation of gametogenesis by described methods (Janse et al. 1985), indicating that these clones are nonproducers in contrast to our earlier report. Generation and selection of karyotype mutants in a high-producer clone. In four
out of eight experiments karyotype mutants replaced the parasites with the original karyotype of clone 8417. Karyotypes of the mutants were different from each other (Fig. 2) and all mutants were nongametocyte producers. In two experiments we were able to follow the overgrowing of the original parasites by the karyotype mutants. Within several weeks the population changed from the original karyotype into the mutant karyotype as shown in Fig. 3. The increase of parasites with the mutant karyotype coincided with the decrease of gametocyte production, suggesting that the loss of gametocyte production and a change in the karyotype occurred simultaneously in the same parasite. Competition between parasites in experimentally mixed infections. We established
six independent mixed infections of a high gametocyte-producer clone (8417) and a nonproducer clone (1) and quantitated rel-
JANSE
1234
1
2
ET AL.
3
4
5 3.5 Mb
0.6 2. Karyotype of four mutants (1-I) which replaced the parent parasites of clone 8417 (5) of Plasmodium berghei in four independent experiments. Major changes of the karyotype are indicated by arrows. FIGE was performed at 3.5 V/cm with linearly increasing forward pulse from 40-350 set and a forward/backward ratio of 3/l. FIG.
ative changes in the proportion of each clone in the mixed populations during 25 weeks of asexual multiplication. Recently we reported detailed comparisons of several growth characteristics of these clones (Janse et al. 1989b). The duration of the schizogonic cycle, reticulocyte preference, and multiplication in viva did not significantly differ between these parasites. The clearly different karyotype allows easy discrimination between these clones in mixed infections (Fig. 4). Chromosome 7 of clone 8417 shows strong hybridization with the 2.3-kb repeat probe (more than 100 repeats; Ponzi et al. 1990), while chromosome 7 of clone 1 does not contain 2.3-kb repeats and is 0.5 megabases smaller. As a result we
were able to detect only 1% parasites of clone 8417 in a serial dilution of these parasites with parasites of clone 1, using FIGE karyotypes hybridized with the 2.3-kb probe (Fig. 5, lanes 3-7). Figure 4 shows the karyotype pattern of the six mixed infections after 1 week of multiplication. The karyotypes of both clones are visible and from the staining intensity of the individual chromosomes it appears that both clones were present in a ratio of 1: 1. In five out of six mixed infections high-producer parasites of clone 8417 were overgrown by nonproducer parasites of clone 1 within 9 to I5 weeks, as was shown by the disappearance of the karyotype of clone 8417. See for example the dis-
P. berghei:
IN VIVO
Al A2
GENERATION
Al
AND
SELECTION
92 91
A2
OF MUTANTS
5
92 91
5 FIG. 3. Karyotype patterns of two populations of clone 8417 of Plasmodium berghei just before (Al, Bl) and just after (A2, B2) the loss of gametocyte production. Hybridization with chromosomespecific probes shows that in population A nonproducer mutants with a smaller chromosome 5 replaced the high-producer parasites of clone 8417. In population B nonproducer mutantswith a smaller chromosome 7 became predominant. Al, Week 48; A2, Week 53; Bl, Week 12; B2, Week 16.
appearance of hybridization of the 2.3-kb probe with chromosome 7 of clone 8417 in two experiments in Fig. 5 (lanes 8-15). The last week in which we could detect parasites of clone 8417 is shown in Table I. In all experiments there was no abrupt change in the proportion of the two clones but the proportion of clone 8417 decreased gradually. As expected, the disappearance of parasites of clone 8417 coincided with loss of gametocyte production and infectivity to mosquitoes (Table I). Mosquitoes, however, still became infected after feeding on mixed infections containing less than 1% parasites of the high-producer clone. In one mixed infection, parasites of the high-producer clone remained present during the period of 25 weeks as was shown by the karyotype pattern of the population
(Fig. 6). Surprisingly, gametocyte production and infectivity to mosquitoes were completely lost after Week 18 in spite of the presence of parasites of clone 8417. This suggests that in this particular infection parasites of clone 8417 had lost the capacity to produce gametocytes and were able to coexist with nonproducer parasites of clone 1. To test this hypothesis we cloned parasites by the method of limiting dilution from the population at Week 25. Out of a total of 25 clones, 7 exhibited the same karyotype as clone 8417 (except for a small change in the size of chromosome 7). All these clones were nonproducers. We followed this mixed infection for another period of 15 weeks in four independent experiments. In two of these experiments nonproducer parasites of clone 8417 predominated the non-
JANSE ET AL.
12345678
8417 ::@I
35Mb
06 4. Karyotype patterns of six mixed infections (lanes 2-7) of clone 8417 (ANKA strain) and clone 1 (K173 strain) of Plasmodium berghei after 1 week of asexual multiplication in mice. Lanes 1 and 8 show the karyotype pattern of experimentally mixed parasites of both clones in a ratio of 1:l). Arrows show the position of chromosome 7 in both clones. FIGE was performed at 3.5 V/cm, with linearly increasing forward pulse from 60-500 set and a forward/backward ratio of 311. Schematic representation of the karyotype of clone 1 and clone 8417 as established by Ponzi et al. (1990). FIG.
1990). The latter process may provide a means for the generation of genetic diversity in addition to gene reassortment and recombination between homologous chromosomes during sexual development of P. DISCUSSION fulciparum (Corcoran et al. 1988; Sinnis In P. berghei karyotype mutants and and Wellems 1988; Vernick et al. 1988). Parasites can readily lose the capacity to nongametocyte producer mutants spontaneously arise and rapidly predominate dur- produce gametocytes during asexual multiing asexual multiplication in vivo (this pa- plication in vivo. We showed here that the per, Janse et al. 1989b). Recently we found decrease of gametocyte production is the not only that karyotype mutants arise as a result of competition between high- and result of deletion of DNA sequences, as nonproducer mutants. Nonproducer mutants rapidly predominated over highwas shown to occur in vitro in P. fulciparum (Corcoran et al. 1988), but also that producer parasites of the parent clone. It recombinational events occur during mito- seems logical that the production of gamesis, resulting in the enlargement of certain tocytes by asexually dividing parasites is a chromosomes (Ponzi et al. 1990; Pace et al. selective disadvantage in terms of overall producers of clone 1, while in the other two experiments parasites of clone 1 became predominant.
P. ber&ei:
12
IN VIVO GENERATION
3 4
5
6
7
7
AND SELECTION OF MUTANTS
8
9 10 11 12 13 14
15
FIG. 5. Karyotype pattern and hybridization pattern with the 2.3-kb probe of mixed infections of clone 8417 and clone 1 of Plasmodium berghei. Arrows show the position of chromosome 7 of both clones. Lane 1, clone 8417; lane 2, clone 1; lanes 3-7, experimentally mixed parasites of clone 8417 and clone 1 in the ratio of 1:l (lane 3), 1:9 (lane 4), 199 (lanes 5,6), and 1:999 (lane 7). Lanes 8-12, mixed infection number 4 at Weeks 4, 8, 10, 12, and 14, respectively. Lanes 13-15: mixed infection number 5 at Weeks 4, 6, and 14, respectively. FIGE was performed at 3.5 V/cm, with linearly increasing forward pulse from 40-350 set and a forward/backward ratio of 3/l.
multiplication rate, since merozoites which develop into gametocytes produce no progeny for the next asexual cycle. We cannot conclude that the difference in gametocyte production is the only factor influencing the competition between the mutants, although it was the only difference detected.
Clones with intermediate gametocyte production were not found in our experiments, suggesting a simple genetic mechanism by which gametocyte production was clones showing a lost. In P. fulciparum, range of different capacities to produce gametocytes have been isolated (Burkot et al.
JANSE
TABLE 1 Disappearance of Gametocyte Production in a Clone of Plasmodium berghei (8417) with a High Capacity of Gametocyte Production Mixed with a Nongametocyte Producing Clone (1) Clone 8417 undetectable from Week” 10 13 13 15 9 clone 8417 remained present
Mosquitoes infected with clone 8417 in Week
Mosquitoes not infected from Week
5, 11 8, 11, 13, 14, 17 8 4, 5, 12, 14 11, 14
14 (4 exp.) 18 (5 exp.) 20 (3 exp.) 20 (4 exp.) 16 (4 exp.)
12, 14
18 (5 exp.)
Nofe. Six mixed infections of these clones were followed over a period of 25 weeks. a This has been established on the basis of the hybridization signal of chromosome 7 of done 8417 with the 2.3-kb probe (see Fig. 5).
1984; Graves et al. 1984) and Said et al. (1986) found P. berghei clones with a very low gametocyte production. In these studies, however, clones were isolated directly from mixed populations rather than being allowed to spontaneously appear as in our 1 2
ET AL.
experiments. It remains to be investigated whether or not intermediate clones exist in our mixed populations. We reported earlier that no clear correlation exists between the karyotype pattern and the capacity to produce gametocytes (Janse et al. 1989b). Although four karyotype mutants described in this paper were nonproducers, suggesting a relationship, we also found nonproducer mutants with no detectable alterations in the karyotype. On the other hand, in two experiments described here the loss of gametocyte production and a change in the karyotype (deletions in chromosomes 7 or 5) occurred simultaneously in the same parasite, which may suggest a causal relationship between the two events. In our experiments, karyotype mutants which do not produce gametocytes are more likely to become dominant in the population in comparison with other karyotype mutants, since nonproducers have a strong selective advantage. Therefore, karyotype mutants with a high gametocyte production will not easily be detected. By cloning parasites from a population of the high-producer clone 8417, we
3456789
FIG. 6. Karyotype pattern and hybridization pattern with the 2.3-kb probe of mixed infection number 6 (clone 8417 and clone 1 of Phmodium berg&i) at Weeks 1, 5, 10, 15, 20, and 25, respectively (lanes l-7). Lane 8, clone 8417; lane 9, clone 1. Arrows show the position of chromosome 7 of both clones.
9
P. berghei: IN VIVO GENERATION AND SELECTION OF MUTANTS
have found karyotype mutants with a high gametocyte production (results not shown), indicating that such mutants do arise but do not become dominant in the population. In nature, the transmission of malaria parasites by their vector mosquitoes is essential for the survival of the parasite. Therefore, one would expect genetic factors controlling the production of gametocytes in Plasmodium to be stable. In P. berghei the gene or set of genes which are responsible for the switch to sexual differentiation are in fact not stable at all. This instability may be explained by the laboratory conditions under which the parasites are maintained in our experiments. However, transmission of malaria parasites in nature is not just a matter of producing as many gametocytes as possible, but is a complex interplay between the parasite and its different hosts. Therefore, selection of a stable and high gametocyte production during prolonged periods of asexual multiplication in the vertebrate host may be absent in nature. Mosquito transmission is a strong selective mechanism, so that only gametocyte-producer parasites will be transmitted to subsequent hosts. Thus, every sporozoite-induced infection will start with an optimal gametocyte production. The process of the loss of gametocyte production we described here resembles the loss of the capacity to cytoadhere in P. fulciparum parasites cultured in vitro. The capacity to cytoadhere is an essential feature in the vertebrate host but can be lost during mitotic multiplication in vitro. Interestingly, it has been found that cytoadherence-negative mutants, which arise during asexual multiplication, were nongametocyte producers and showed a deletion on chromosome 9 (Forsyth et al. 1990; K. P. Forsyth, personal communication). In the light of the observations described here it would be very interesting to know if there is a causal relationship between this change of the karyotype, the loss of cytoadherence, and the loss of gametocyte production.
ACKNOWLEDGMENT This work was carried out under EEC contract TS20126-l(NL) in the framework of the program “Science and Technology for Development.” REFERENCES V. K., AND TRACER, W. 1984. Gametocyte forming and nongametocyte forming clones of P/as-
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Received 27 November 1990; accepted with revision 20 July 1991
