InrernmionalJoumal Jor Parasitology Printed in Grew Britain
Vol. 20. NO. 5,pp. 61S618,
1990 0
002~7519/90 $3.00 + 0.00 Pergamon Press p/c Society for Pnrasimlogy
1990 Aurtr&m
GAMETOCYTOGENESIS INDUCTION BY AMMONIUM COMPOUNDS IN CULTURED PLASMODIUM FALCIPARUM* T. ONO~
and T. NAKABAYASHI~
Department of Protozoology, Research Institute for Microbial Diseases, Osaka University, Suita Osaka 565, Japan (Received 13 November 1989; accepted 5 February 1990)
Abstract~No T. and NAKABAYASHIT. 1990. Gametocytogenesis induction by ammonium compounds in cultured Plasmodium falciparum. International Journalfor Parasitology 20: 615418. Asexual parasites of a strain that seldom or never produce gametocytes in in vitro culture began gametocytogenesis after 24 h treatment with RPM1 1640 medium containing concanavalin A (final concentration, 10 pg ml-‘) and ammonium carbonate (final concentration, 15 mM ml-‘) or ammonium bicarbonate (final concentration, 15 mMml-‘). Gametocytogenesis was consistently observed from the 3rd day after the treatment. Concanavalin A enhanced gametocytogenesis induction by ammonium carbonate or ammonium bicarbonate, although concanavalin A does not itself have gametocytogenesis induction activity. Whereas no gametocytogenesis was observed after addition of concanavalin A and ammonium acetate (final concentration, 5-25 mM ml-‘) or ammonium chloride (final concentration, 5-15 mM_‘). Addition of ammonium compounds resulted in decrease of parasitemia, regardless of gametocytogenesis. INDEX KEY WORDS: Gametocytogenesis induction; Plasmodium falciparum; ammonium compounds; concanavalin A.
INTRODUCTION THE
induction of gametocytogenesis is important not only for the biological characterization of gametocytogenesis but also as a starting point for an in vitro system for the production of sporozoites. Until now, however, many attempts to induce gametocytogenesis in cultured parasites have been unsuccessful. Therefore, studies on gametocytes and gametocytogenesis have been carried out on cultured strains that spontaneously produce many gametocytes (Carter & Beach, 1977; Kaushal, Carter, Miller & Krishna, 1980; Ponnudurai, Lensen, Leeuwenberg & Meuwissen, 1982; Carter, Suhrbier, Beckers & Sinden, 1987). In previous reports (Ono, Nakai & Nakabayashi, 1986; Ono & Nakabayashi, 1989), we found that asexual parasites of some strains which seldom or never produce gametocytes began gametocytogenesis after treatment with RPM1 1640 medium, prepared by dissolving powdered RPM1 1640 medium in culture supernatant of anti-P. falciparum antibody producing * This work was presented in part at the 7th JapaneseGerman Cooperative Symposium on Protozoan Diseases, Tokushima, Japan, 19-23 July 1989. t Present address and address for correspondence: Department of Veterinary Epidemiology, College of Agriculture, University of Osaka Prefecture, Mozu-Umemachi, Sakai, Osaka 591, Japan. $ Present address: Department of Parasitology, School of Medicine, Fujita-Gakuen Health University, KutsukakeCho, Toyoake 470- 11, Japan.
hybridoma cells, in the place of distilled water. In the present study, we observed that asexual parasites also began gametocytogenesis following 24 h treatment with RPM1 1640 medium containing ammonium carbonate or ammonium bicarbonate. MATERIALS AND METHODS Strain of Plasmodium
falciparum. P. falciparum strain Falciparum-Vietnam-Oaknoll (FVO) used in this study was provided by courtesy of Dr W. A. Siddiqui, Department of Tropical Medicine, University of Hawaii. This strain was the same as that used in our previous reports (Ono et al., 1986; Ono & Nakabayashi, 1989). Strain FVO seldom or never produces gametocytes in culture with RPM1 1640 medium. Culture method. Parasites were cultured by the method of Siddiqui (1979) in 100 ml Erlenmeyer flasks. The flasks were fitted with stoppers, with entry parts for a gas mixture of 90% N,, 8% CO, and 2% 0,. A gas mixture constantly passed through each flask. The RPM1 1640 medium was used as culture medium in the present study. The culture medium (OH 7.45) consisting of the RPM1 1640 medium (GIBCO). 25 mM-HEPES, 24 mM-sodium bicarbonate, ~3.4 mM-Lglutamine and 50 mhi-hypoxanthine was prepared every 3-4 weeks, stored at 4“C, and warmed to 37’C prior to use. Type 0 blood was used in the present experiment. Type 0 blood was drawn into a syringe containing CPD (sodium citrate, citric acid, sodium phosphate, dibasic, 2-hydrate and dextrose) solution as the anticoagulant. A mixture of 200 ml of blood and 28 ml of CPD solution was stored at 4°C and was used within 3 weeks. Erythrocytes were washed five times with RPM1 1640 medium by centrifugation at 750 g for 10 min. After the first washing, the huffy coat was removed. After the fifth washing, the pellet of packed cells was 615
616
T. ONO and T NAKABAYASHI
resuspended in an equal volume of RPM1 1640 medium. Infected erythrocytes were added to fresh erythrocytes to make up a total volume of I ml with a parasitemia of approximately 0.5%. One millilitre of suspension of infected erythrocytes was inoculated into medium containing 8 ml of RPM1 1640 medium and 1 ml of horse serum inactivated by heating at 56’C for 30 min and incubated at 37°C. The medium was changed after 48 h culture. After that, the medium was renewed every 24 h. Horse serum was not absorbed with erythrocytes prior to use. On day 5 of culture in regular RPM1 1640 medium containing 10% horse serum, when parasitemia reached 3-5%, the medium was replaced by special RPM! 1640 medium containing various ammonium compounds (Wako pure chemicals), concanavalin A (final concentration 10 pg ml- ‘, Sigma Chemical Company) and horse serum. After culturing for 24 h, the medium was changed to regular RPM1 1640 medium containing 10% horse serum. Addition of 10 pg of concanavalin A to the parasite culture in RPM1 1640 medium with or without ammonium compounds did not result in agglutination of erythrocytes. In the present experiment, various ammonium compounds and concanavalin A were adjusted to pH 7.4 prior to use. When special RPM1 1640 media containing various ammonium compounds and 10% horse serum were used alone or in combination with IO pg ml- ’ concanavalin A for 24 h, the pH of the culture media was 7.4-7.5. It was the same pH as in RPM1 1640 medium prior to use. After that, when regular RPM1 1640 medium containing 10% horse
serum was used, the culture medium was pH 7.2-7.3. Thereafter, culture with regular RPM1 1640 medium maintained a pH of 7.2-7.3. The induction of gametocytogenesis was judged by examination of Giemsa-stained blood smears. The stages of gametocytes found in smears were classified on the basis of the standard reported by Hawking, Wilson & Ganmage (1971) and Carter & Miller (1979). However, stage I gametocytes are difficult to distinguish exactly from asexual trophozoites; thus, in the present experiment gametocytes were classified into four groups: stages II, III, IV and V. RESULTS
Gametocytogenesis was consistently observed from the 3rd day after the addition of ammonium carbonate (final concentration, 15 mM) or ammonium bicarbonate (final concentration, 15 mM) with or without concanavalin A, as shown in Table 1. Addition of 10 pg of concanavalin A to special RPM1 1640 medium containing ammonium carbonate or ammonium bicarbonate enhanced the induction of gametocytogenesis. Figure 1 ad showed gametocytes appearing 5 days after the treatment with ammonium bicarbonate and concanavalin A. After the treatment with ammonium compounds gametocytes were seen until the 8th day in regular RPM1 1640 medium. The
TABLE ~-INDUCTION OF GAMETOCYTOGENESIS BY AMMONIUMCOMPOUNDS*AND CONCANAVAL~N At IN CULTURED Plasmodium falciparum; INDucEa 0F ciAh4ETocYToGENEslsANDCONCANAVALIN A WEREADDED0~ DAY 5 0~ PARASITECULTURE Inducer
of gametocytogenesis
Days after addition
of inducer
of gametocytogenesis$
Con At O$l
Ammonium
carbonate
-
Ammonium
carbonate
+
Ammonium
Ammonium
bicarbonate
bicarbonate
_
+
_
+
4.2 0 0 0 4.5 0 0 0 3.8 0 0 0 4.0 0 0 0 4.1 0 0 0 3.7 0 0 0
23456789 2.8 0 0 0 4.0 0 0 0 1.8 0 0 0 3.5 0 0 0 8.1 0 0 0 7.8 0 0 0
1.9 0 0 0000 2.5
I 0 0 1.2 0 0 0 2.0 1 0 00 12.3 0 0 0 11.7 0 0 0
1.5 13 3
1.3 16 2
1.3 13 6
1.0 22 4 0 1.3 10 17 0 1.4 22 16561
1.3 28 6 13 1.3 12
0.9 29 12
10.0 0 0 0 8.7 0 0 0
12 1.1 26 1113 9.0 0 0 0 8.1 10 0 0
1.5 8 5 1.3 23
8.3 0 0 0 8.0 0 0
1.2 8 43 10 1.3 26 9 20 1.0 12 74 20 0.6 21
5.9 00 00 00 6.7 00 00 00
0.8 0
1.0 3 6 0.9 5
0.7 7
6.5
6.0
1.0 2 3 0 1.0 1 2 1 0.7 2 1 0 1.1 1 3 1 6.5 0 0 0 5.5 0 0 0
0.811
Ori 0** Ott 1.2 0 0 1 1.0 0 0 0 1.0 0 0 0 5.0 0 0 0 6.4 0 0 0
* Final concentration, 15 mM ml-l. t Concanavalin A, final concentration 10 pg ml-‘. $Each figure represents the mean of eight replicate experiments. §Ammonium compounds and concanavalin A were added on this day. lIThe first line shows parasitemia as the percentage of parasites in 400 erythrocytes examined. TThe second line shows the number of stage II gametocytes among 300 parasites. **The third line shows the number of stage III gametocytes among 300 parasites. ttThe fourth line shows the number of stage IV gametocytes among 300 parasites.
617
Gametocytogenesis in cultured P. falciparum
FIG.
1. (a), (b), (c) and (d) show gametocytes which appeared 5 days after the treatment with ammonium bicarbonate (final concentration, 15 mM ml-‘) and concanavalin A (final concentration, 10 fig ml-‘). Scale bar = 3pm. (a) shows stage II gametocyte. (b) and (c) show stage III gametocytes. (d) shows stage V gametocyte which can be seen only rarely.
number of gametocytes is higher between the 3rd and 6th days after treatment. When regular RPM1 1640 medium with concanavalin A was used, no gametocytogenesis was observed, as in regular RPM1 1640 medium alone. For analysis of the interaction between the induction of gametocytogenesis and the rate of appearance of parasites, parasitemia was examined daily. When parasite culture was started in regular RPM1 1640 medium, parasitemia increased day by day. As shown in Table 1, however, when special RPM1 1640 medium containing ammonium carbonate or ammonium bicarbonate with or without concanavalin A was used for 24 h on day 5 of culture with regular RPM1 1640 medium, the number of parasites decreased. No increase in parasitemia was seen throughout the observation period. Although not included in the table, no gametocytogenesis was observed after addition of ammonium acetate (final concentrations 5-25 mM) or ammonium chloride (final concentrations S-15 mM>with or without concanavalin A. Addition of these ammonium compounds, which have no ability to induce gametocytogenesis, also results in a decrease in parasitemia. The number of parasites did not increase throughout the observation period. DISCUSSION Conversion of asexual parasites to gametocytes occurs not only during natural infections but also in
continuous
in vitro
culture (Trager & Jensen, 1976;
Jensen, 1979). Kaushal et al. (1980) reported that conversion of asexual parasites to gametocytes was enhanced in static culture with 1 mr+cyclic AMP. However, Brockelman (1982) and Inselburg (1983) have unsuccessfully tried to confirm the gametocytogenesis-inducing effect of cyclic AMP. We have also failed to induce gametocytogenesis by exposing parasite culture to cyclic AMP (unpublished data). In previous reports (Ono et al., 1986; Ono & Nakabayashi, 1989), we found that gametocytogenesis was induced by a special RPM1 1640 medium prepared with culture supernatants from hybridoma cells of two hybrid lines that produce anti-P. falciparum antibody. In the present study, we found that gametocytogenesis was also induced in parasites culture in RPM1 1640 medium containing ammonium carbonate or ammonium bicarbonate. Gametocytogenesis induction with the known chemical substances appears to be more convenient than that with culture supernatant of hybridoma cells used in the previous reports (Ono et al., 1986; Ono & Nakabayashi, 1989) to analyze mechanism of gametocytogenesis. In our previous report (Ono et al., 1986), we observed that the addition of 10 pugml-’ concanavalin A (final concentration) to a special medium, prepared with culture supernatant from hybridoma cells of two hybrid lines that produce anti-P. falciparum antibody, enhanced the induction of gametocytogenesis. Therefore, in the present study the induction of gametocytogenesis was carried out using ammonium compounds with concanavalin A. Concanavalin A does not itself have gametocytogenesis induction activity, as shown in the table. But, concanavalin A enhanced the rate of gametocytogenesis induction in the induction with ammonium compounds as well as in that with culture supernatants of hybridoma cells. The mechanism of the effect of concanavalin A on gametocytogenesis is not known. In the present study addition of ammonium carbonate or ammonium bicarbonate to parasite cultures results in a decrease of parasitemia. The addition of ammonium acetate or ammonium chloride, which have no ability to induce gametocytogenesis, also decreases parasitemias. No direct relation between induction of gametocytogenesis and decrease of parasitemia was demonstrated. It means that environmental conditions which only have adverse effects on the growth of asexual parasites in culture are not a trigger for gametocytogenesis. In the present experiments, gametocytes were usually seen for 6 days only. Therefore, prolonged culture of gametocytes that appear after treatment with ammonium carbonate or ammonium bicarbonate is now being tried by a modification of the culture method. REFERENCES BR~CKELMAN C.
R. 1982. Conditions favouring gametocytogenesis in the continuous culture of Plasmodium falci-
pawn. Journal of Parasitology 29: 454-458. CARTERE. H., SUHRBIER A., BECKERSP. J. A. & SINDENR. E. 1987. The in vitro cultivation of P. falciparum ookinetes
618
T. ONO and T. NAKABAYASHI
and their enrichment on Nycodenz” density gradients. Parasitology 95: 25-30. CARTERR. & BEACHR. F. 1977. Gametogenesis in culture by gametocytes of Plasmodium falciparum. Nature (London) 270: 240-24 1. CARTERR. & MILLERL. H. 1979. Evidence for environmental modulation of gametocytogenesis in Plasmodium falciparum in continuous culture. Bulletin of the World Health Organization 57 (Suppl.): 37-52. HAWKINGF., WILSONM. E. & GANMAGE K. 1971. Evidence for cyclic development and short-lived maturity in the gametocytes of Plasmodiumfalciparum. Transactions of the Royal Society of Tropical Medicine and Hygiene 65: 549559. INSELBURGJ. 1983. Gametocyte formation by the progeny of single Plasmodium falciparum schizonts. Journal of Parasitology 69: 584-591. JENSEN J. B. 1979. Observations on gametogenesis in Plasmodium falciparum from continuous cultures. Journal of Protozoology 26: 129-132. KAUSHAL D. C., CARTER R., MILLER L. H. & KRISHNA G. 1980. Gametocytogenesis by malaria parasites in con-
tinuous culture. Nature (London) 286: 49&492. ONO T., NAKAI & NAKA~AYASHI T. 1986. Induction of gametocytogenesis in Plasmodium falciparum by the culture supernatant of hybridoma cells producing anti-P. falciparum antibody. Biken Journal 29: 77-81. ONO T. & NAKABAYASHI T. 1989. Gametocytogenesis induction in cultured Plasmodium falciparum and further development of the gametocytes to ookinetes in prolonged culture. Parasitology Research 75: 189-193. PONNUDURAIT., LENSENA. H. W., LEEUWENBERG A. D. E. M. & MEUWISSENJ. H. E. TH. 1982. Cultivation of fertile Plasmodium falciparum gametocytes in semi-automated systems. 1. Static cultures. Transactions of fhe Royal Society of Tropical Medicine and Hygiene 76: 8 12-8 18. SIDDIQUI W. A. 1979. Continuous in vitro cultivation of Plasmodium falciparum in human erythrocytes: description of a single technique to obtain high yields of parasites. In: Practical Tissue Culture Applications (Edited by MARMORCH K. & HIRUMI H.), pp. 267-277. Academic Press, New York. TRACER W. &JENSEN J. B. 1976. Human malaria parasites in continuous culture. Science 193: 673-675.
Vol. 20. NO. 5,pp. 61S618,
1990 0
002~7519/90 $3.00 + 0.00 Pergamon Press p/c Society for Pnrasimlogy
1990 Aurtr&m
GAMETOCYTOGENESIS INDUCTION BY AMMONIUM COMPOUNDS IN CULTURED PLASMODIUM FALCIPARUM* T. ONO~
and T. NAKABAYASHI~
Department of Protozoology, Research Institute for Microbial Diseases, Osaka University, Suita Osaka 565, Japan (Received 13 November 1989; accepted 5 February 1990)
Abstract~No T. and NAKABAYASHIT. 1990. Gametocytogenesis induction by ammonium compounds in cultured Plasmodium falciparum. International Journalfor Parasitology 20: 615418. Asexual parasites of a strain that seldom or never produce gametocytes in in vitro culture began gametocytogenesis after 24 h treatment with RPM1 1640 medium containing concanavalin A (final concentration, 10 pg ml-‘) and ammonium carbonate (final concentration, 15 mM ml-‘) or ammonium bicarbonate (final concentration, 15 mMml-‘). Gametocytogenesis was consistently observed from the 3rd day after the treatment. Concanavalin A enhanced gametocytogenesis induction by ammonium carbonate or ammonium bicarbonate, although concanavalin A does not itself have gametocytogenesis induction activity. Whereas no gametocytogenesis was observed after addition of concanavalin A and ammonium acetate (final concentration, 5-25 mM ml-‘) or ammonium chloride (final concentration, 5-15 mM_‘). Addition of ammonium compounds resulted in decrease of parasitemia, regardless of gametocytogenesis. INDEX KEY WORDS: Gametocytogenesis induction; Plasmodium falciparum; ammonium compounds; concanavalin A.
INTRODUCTION THE
induction of gametocytogenesis is important not only for the biological characterization of gametocytogenesis but also as a starting point for an in vitro system for the production of sporozoites. Until now, however, many attempts to induce gametocytogenesis in cultured parasites have been unsuccessful. Therefore, studies on gametocytes and gametocytogenesis have been carried out on cultured strains that spontaneously produce many gametocytes (Carter & Beach, 1977; Kaushal, Carter, Miller & Krishna, 1980; Ponnudurai, Lensen, Leeuwenberg & Meuwissen, 1982; Carter, Suhrbier, Beckers & Sinden, 1987). In previous reports (Ono, Nakai & Nakabayashi, 1986; Ono & Nakabayashi, 1989), we found that asexual parasites of some strains which seldom or never produce gametocytes began gametocytogenesis after treatment with RPM1 1640 medium, prepared by dissolving powdered RPM1 1640 medium in culture supernatant of anti-P. falciparum antibody producing * This work was presented in part at the 7th JapaneseGerman Cooperative Symposium on Protozoan Diseases, Tokushima, Japan, 19-23 July 1989. t Present address and address for correspondence: Department of Veterinary Epidemiology, College of Agriculture, University of Osaka Prefecture, Mozu-Umemachi, Sakai, Osaka 591, Japan. $ Present address: Department of Parasitology, School of Medicine, Fujita-Gakuen Health University, KutsukakeCho, Toyoake 470- 11, Japan.
hybridoma cells, in the place of distilled water. In the present study, we observed that asexual parasites also began gametocytogenesis following 24 h treatment with RPM1 1640 medium containing ammonium carbonate or ammonium bicarbonate. MATERIALS AND METHODS Strain of Plasmodium
falciparum. P. falciparum strain Falciparum-Vietnam-Oaknoll (FVO) used in this study was provided by courtesy of Dr W. A. Siddiqui, Department of Tropical Medicine, University of Hawaii. This strain was the same as that used in our previous reports (Ono et al., 1986; Ono & Nakabayashi, 1989). Strain FVO seldom or never produces gametocytes in culture with RPM1 1640 medium. Culture method. Parasites were cultured by the method of Siddiqui (1979) in 100 ml Erlenmeyer flasks. The flasks were fitted with stoppers, with entry parts for a gas mixture of 90% N,, 8% CO, and 2% 0,. A gas mixture constantly passed through each flask. The RPM1 1640 medium was used as culture medium in the present study. The culture medium (OH 7.45) consisting of the RPM1 1640 medium (GIBCO). 25 mM-HEPES, 24 mM-sodium bicarbonate, ~3.4 mM-Lglutamine and 50 mhi-hypoxanthine was prepared every 3-4 weeks, stored at 4“C, and warmed to 37’C prior to use. Type 0 blood was used in the present experiment. Type 0 blood was drawn into a syringe containing CPD (sodium citrate, citric acid, sodium phosphate, dibasic, 2-hydrate and dextrose) solution as the anticoagulant. A mixture of 200 ml of blood and 28 ml of CPD solution was stored at 4°C and was used within 3 weeks. Erythrocytes were washed five times with RPM1 1640 medium by centrifugation at 750 g for 10 min. After the first washing, the huffy coat was removed. After the fifth washing, the pellet of packed cells was 615
616
T. ONO and T NAKABAYASHI
resuspended in an equal volume of RPM1 1640 medium. Infected erythrocytes were added to fresh erythrocytes to make up a total volume of I ml with a parasitemia of approximately 0.5%. One millilitre of suspension of infected erythrocytes was inoculated into medium containing 8 ml of RPM1 1640 medium and 1 ml of horse serum inactivated by heating at 56’C for 30 min and incubated at 37°C. The medium was changed after 48 h culture. After that, the medium was renewed every 24 h. Horse serum was not absorbed with erythrocytes prior to use. On day 5 of culture in regular RPM1 1640 medium containing 10% horse serum, when parasitemia reached 3-5%, the medium was replaced by special RPM! 1640 medium containing various ammonium compounds (Wako pure chemicals), concanavalin A (final concentration 10 pg ml- ‘, Sigma Chemical Company) and horse serum. After culturing for 24 h, the medium was changed to regular RPM1 1640 medium containing 10% horse serum. Addition of 10 pg of concanavalin A to the parasite culture in RPM1 1640 medium with or without ammonium compounds did not result in agglutination of erythrocytes. In the present experiment, various ammonium compounds and concanavalin A were adjusted to pH 7.4 prior to use. When special RPM1 1640 media containing various ammonium compounds and 10% horse serum were used alone or in combination with IO pg ml- ’ concanavalin A for 24 h, the pH of the culture media was 7.4-7.5. It was the same pH as in RPM1 1640 medium prior to use. After that, when regular RPM1 1640 medium containing 10% horse
serum was used, the culture medium was pH 7.2-7.3. Thereafter, culture with regular RPM1 1640 medium maintained a pH of 7.2-7.3. The induction of gametocytogenesis was judged by examination of Giemsa-stained blood smears. The stages of gametocytes found in smears were classified on the basis of the standard reported by Hawking, Wilson & Ganmage (1971) and Carter & Miller (1979). However, stage I gametocytes are difficult to distinguish exactly from asexual trophozoites; thus, in the present experiment gametocytes were classified into four groups: stages II, III, IV and V. RESULTS
Gametocytogenesis was consistently observed from the 3rd day after the addition of ammonium carbonate (final concentration, 15 mM) or ammonium bicarbonate (final concentration, 15 mM) with or without concanavalin A, as shown in Table 1. Addition of 10 pg of concanavalin A to special RPM1 1640 medium containing ammonium carbonate or ammonium bicarbonate enhanced the induction of gametocytogenesis. Figure 1 ad showed gametocytes appearing 5 days after the treatment with ammonium bicarbonate and concanavalin A. After the treatment with ammonium compounds gametocytes were seen until the 8th day in regular RPM1 1640 medium. The
TABLE ~-INDUCTION OF GAMETOCYTOGENESIS BY AMMONIUMCOMPOUNDS*AND CONCANAVAL~N At IN CULTURED Plasmodium falciparum; INDucEa 0F ciAh4ETocYToGENEslsANDCONCANAVALIN A WEREADDED0~ DAY 5 0~ PARASITECULTURE Inducer
of gametocytogenesis
Days after addition
of inducer
of gametocytogenesis$
Con At O$l
Ammonium
carbonate
-
Ammonium
carbonate
+
Ammonium
Ammonium
bicarbonate
bicarbonate
_
+
_
+
4.2 0 0 0 4.5 0 0 0 3.8 0 0 0 4.0 0 0 0 4.1 0 0 0 3.7 0 0 0
23456789 2.8 0 0 0 4.0 0 0 0 1.8 0 0 0 3.5 0 0 0 8.1 0 0 0 7.8 0 0 0
1.9 0 0 0000 2.5
I 0 0 1.2 0 0 0 2.0 1 0 00 12.3 0 0 0 11.7 0 0 0
1.5 13 3
1.3 16 2
1.3 13 6
1.0 22 4 0 1.3 10 17 0 1.4 22 16561
1.3 28 6 13 1.3 12
0.9 29 12
10.0 0 0 0 8.7 0 0 0
12 1.1 26 1113 9.0 0 0 0 8.1 10 0 0
1.5 8 5 1.3 23
8.3 0 0 0 8.0 0 0
1.2 8 43 10 1.3 26 9 20 1.0 12 74 20 0.6 21
5.9 00 00 00 6.7 00 00 00
0.8 0
1.0 3 6 0.9 5
0.7 7
6.5
6.0
1.0 2 3 0 1.0 1 2 1 0.7 2 1 0 1.1 1 3 1 6.5 0 0 0 5.5 0 0 0
0.811
Ori 0** Ott 1.2 0 0 1 1.0 0 0 0 1.0 0 0 0 5.0 0 0 0 6.4 0 0 0
* Final concentration, 15 mM ml-l. t Concanavalin A, final concentration 10 pg ml-‘. $Each figure represents the mean of eight replicate experiments. §Ammonium compounds and concanavalin A were added on this day. lIThe first line shows parasitemia as the percentage of parasites in 400 erythrocytes examined. TThe second line shows the number of stage II gametocytes among 300 parasites. **The third line shows the number of stage III gametocytes among 300 parasites. ttThe fourth line shows the number of stage IV gametocytes among 300 parasites.
617
Gametocytogenesis in cultured P. falciparum
FIG.
1. (a), (b), (c) and (d) show gametocytes which appeared 5 days after the treatment with ammonium bicarbonate (final concentration, 15 mM ml-‘) and concanavalin A (final concentration, 10 fig ml-‘). Scale bar = 3pm. (a) shows stage II gametocyte. (b) and (c) show stage III gametocytes. (d) shows stage V gametocyte which can be seen only rarely.
number of gametocytes is higher between the 3rd and 6th days after treatment. When regular RPM1 1640 medium with concanavalin A was used, no gametocytogenesis was observed, as in regular RPM1 1640 medium alone. For analysis of the interaction between the induction of gametocytogenesis and the rate of appearance of parasites, parasitemia was examined daily. When parasite culture was started in regular RPM1 1640 medium, parasitemia increased day by day. As shown in Table 1, however, when special RPM1 1640 medium containing ammonium carbonate or ammonium bicarbonate with or without concanavalin A was used for 24 h on day 5 of culture with regular RPM1 1640 medium, the number of parasites decreased. No increase in parasitemia was seen throughout the observation period. Although not included in the table, no gametocytogenesis was observed after addition of ammonium acetate (final concentrations 5-25 mM) or ammonium chloride (final concentrations S-15 mM>with or without concanavalin A. Addition of these ammonium compounds, which have no ability to induce gametocytogenesis, also results in a decrease in parasitemia. The number of parasites did not increase throughout the observation period. DISCUSSION Conversion of asexual parasites to gametocytes occurs not only during natural infections but also in
continuous
in vitro
culture (Trager & Jensen, 1976;
Jensen, 1979). Kaushal et al. (1980) reported that conversion of asexual parasites to gametocytes was enhanced in static culture with 1 mr+cyclic AMP. However, Brockelman (1982) and Inselburg (1983) have unsuccessfully tried to confirm the gametocytogenesis-inducing effect of cyclic AMP. We have also failed to induce gametocytogenesis by exposing parasite culture to cyclic AMP (unpublished data). In previous reports (Ono et al., 1986; Ono & Nakabayashi, 1989), we found that gametocytogenesis was induced by a special RPM1 1640 medium prepared with culture supernatants from hybridoma cells of two hybrid lines that produce anti-P. falciparum antibody. In the present study, we found that gametocytogenesis was also induced in parasites culture in RPM1 1640 medium containing ammonium carbonate or ammonium bicarbonate. Gametocytogenesis induction with the known chemical substances appears to be more convenient than that with culture supernatant of hybridoma cells used in the previous reports (Ono et al., 1986; Ono & Nakabayashi, 1989) to analyze mechanism of gametocytogenesis. In our previous report (Ono et al., 1986), we observed that the addition of 10 pugml-’ concanavalin A (final concentration) to a special medium, prepared with culture supernatant from hybridoma cells of two hybrid lines that produce anti-P. falciparum antibody, enhanced the induction of gametocytogenesis. Therefore, in the present study the induction of gametocytogenesis was carried out using ammonium compounds with concanavalin A. Concanavalin A does not itself have gametocytogenesis induction activity, as shown in the table. But, concanavalin A enhanced the rate of gametocytogenesis induction in the induction with ammonium compounds as well as in that with culture supernatants of hybridoma cells. The mechanism of the effect of concanavalin A on gametocytogenesis is not known. In the present study addition of ammonium carbonate or ammonium bicarbonate to parasite cultures results in a decrease of parasitemia. The addition of ammonium acetate or ammonium chloride, which have no ability to induce gametocytogenesis, also decreases parasitemias. No direct relation between induction of gametocytogenesis and decrease of parasitemia was demonstrated. It means that environmental conditions which only have adverse effects on the growth of asexual parasites in culture are not a trigger for gametocytogenesis. In the present experiments, gametocytes were usually seen for 6 days only. Therefore, prolonged culture of gametocytes that appear after treatment with ammonium carbonate or ammonium bicarbonate is now being tried by a modification of the culture method. REFERENCES BR~CKELMAN C.
R. 1982. Conditions favouring gametocytogenesis in the continuous culture of Plasmodium falci-
pawn. Journal of Parasitology 29: 454-458. CARTERE. H., SUHRBIER A., BECKERSP. J. A. & SINDENR. E. 1987. The in vitro cultivation of P. falciparum ookinetes
618
T. ONO and T. NAKABAYASHI
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