EXPERIMENTAL
39,
PARASITOLOGY
Use of Amino
Department
of
115-118
Acids
(19%)
by Plasmodium
relicturn
GORDON H. BALLANDJOWETT CHAO Biology, University of California, Los Angeles, (Accepted
for publication
May
Oocysts
California
in Vitro
90024
12, 1975)
BALL, G. H., AND CHAO, J. 1976. Use of amino acids by Plasmodium relicturn oocysts in vitro. Experimental Parasitology 39, 115-118. A comparison was made of the in vitro growth of the gut of Culex tarsalis in Grace’s insect culture medium, supplemented with fetal bovine serum in the presence of dividing cells of Antheraea eucalypti, with a similar preparation of a gut infected with oocysts of the avian parasite, Plasmodium relicturn. In the latter case, after 16 hr, significant decreases occurred in the concentration of arginine, asparagine, and glutamine combined, glutamic acid, glycine, histidine, lysine, proline, and serine. Lower and less marked decreased concentrations of alanine, p-alanine, cystine, isoleucine, leucine, methionine, ornithine, phenylalanine, threonine, tryptophane, tyrosine, and valine also took place. This indicated utilization of certain amino acids by the developing oocysts of P. relicturn in the presence of metabolizing insect cells. INDEX DESCRIPTORS: Plasmodium relicturn; Cubx tarsalis; Tissue culture; Amino acids.
INTRODUCTION
In the formulation of media for the cultivation of insect cells, the composition at first was largely empirical. Media proved successful for growing other tissues in vitro were employed, and it was found necessary to supplement these with insect hemolymph, usually of the same or of a related species (Grace 1962, 1966). Later it was shown that the hemolymph, often difficult to obtain or to keep unchanged, could be replaced by fetal bovine serum (Yunker, Vaughn, and Cory 1967, Hsu, Liu, and Suitor 1969, Sohi 1969). Grace and Brzostowski (1966) demonstrated that the cells of the moth, Antheraea eucalypti used 14 of 21 amino acids over 7 days culture while the concentration of seven others either increased or remained unchanged. Hayashi and Sohi (1970) showed that cells of Aedes aegypti ( Antheraea eucalypti?) incorporated leutine, the only amino acid tested, over a 5-hr period. Igarashi et al. (1973) found 115 Copyright All rights
1976 by Academic Press, Inc. o3 reproduction in any form reserved.
that “nonessential” amino acids stimulated growth over a 7-day period of Aedes albopictus cells. Landureau and Joll&s (1969) showed that 15 amino acids were required for optimum growth and transfer of cells of embryonic Periplaneta americana tissue followed for 7 days. Stanley (1972) summarized conditions for cultivating arthropod cells, including the conflicting results of various investigators on the utilization of amino acids. To the best of our knowledge, no such determination has been made for malarial oocysts developing in insect cell culture. MATERIALS
AND
METHODS
One Culex tars&s stomach, either noninfected or infected, was added in each experiment to 1 ml of medium, containing 1 X lo6 cells. The development of oocysts of PZasmodium relicturn attached to the midgut of Culex tarsalis and cultured in the presence of Grace’s insect cells in Grace’s medium
116
BALL
AND
containing fetal bovine serum has been described previously (Ball and Chao 1971). This medium was supplemented with 100 units of penicillin and 0.1 mg of streptomycin/ml. This cell line, originally believed to be that of Aedes aegypti, was found to be one of Antheraea eucalypti (Greene et al. 1972). Oocyst development was more favorable in this medium than in the medium without A. eucalypti cells or than in Singh’s medium containing cells of A. aegypti (Ball and Chao 1971). The amino acids in the culture medium were analyzed by a Beckman Model 120 amino acid analyzer. Samples were 50 ~1 in volume. Determinations were made of the medium alone at the start of the run, the medium containing an uninfected C. tarsalis stomach and A. eucalypti cells after 16 hr, and the medium containing a C. tarsalis stomach with attached mature oocysts of P. relictum and A. eucalypti cells after I6 hr. All tabulations shown are in millimoles/1000 ml of medium. Cultures were maintained at 22-23 C. The analysis of the medium that contained infected stomachs and A. eucalypti cells was the average of determinations of two parallel cultures. The inherent error of the analyzer decreased in significance as the differences in concentrations became greater. The reliability of the readings as determined by the operator were for differences of less than 0.2 m-moles, more than 20%; for 0.2-0.6 mmoles, *lo-20%; for 0.6-1.0 mmoles, *5-10%; for 1.0-2.0 mmoles, approximately *5%, and for more than 2.0 mmoles, 23-5s. A difference was not considered significant unless it was at least three times this inherent error. RESULTS
Over a 16-hr period, Grace’s medium containing growing cells of Antheraea eucalypti and a stomach of the mosquito Culex tarsalis showed an increase in the concentration of amino acids and of ammonia. On the other hand, if the mosquito
CHAO
stomach contained mature oocysts of Pla.smodium relicturn at the time it was added to the culture, the concentration of amino acids decreased over the same period of time in every case except for aspartic acid, which showed a small increase. The extent of decrease in amino acids of the infected stomach over the uninfected one in the presence of Antheraea eucalypti cells is indicated in Table I. Those amino acids which showed a significant decrease on the basis of our criteria in 16 hr of cultivation were arginine, asparagine, and glutamine (not separable), glutamic acid, glycine, histidine, lysine, proline, and serine. Lower but less marked decreases occurred in the concentrations of alanine, p-alanine, cystine, isoleucine, leucine, methionine, ornithine, phenylalanine, threonine, tryptophane, tyrosine, and valine. The increased concentration of aspartic acid was not significant. Ammonia, on the other hand, showed a significant increase. DISCUSSION
The increase shown in the concentration of amino acids over a I6-hr period in the media containing an uninfected stomach of Culex tarsalis and metabolizing cells of Antheraea eucalypti was probabIy due to the breakdown of some types of mosquito cells which did not survive in vitro. The method of amino acid assay documented in Table I did not indicate any transaminations or use of breakdown products by any amino acid. If these processes did occur, actual utilization of various amino acids by the oocysts would be greater than that recorded. Landureau and Jolles (1969) pointed out the evidence for transamination taking place in their cell cultures of Periplaneta americana. The large increase in the concentration of ammonia may indicate increased deamination activity at the level of various amino acids (Gerard and Gilles 1972). The only difference between the two media assayed for amino acids after 16 hr
AMINO
ACID
METABOLISM
IN
TABLE Concentration Amino
of Amino
acids
Larginine baspartic acid basparagine, L-glutamine (not separable) Lalanine p-&nine 4 cystine L-glut,amic acid 1,glutamine Glgcine I.-hi&dine r,isoleucine I,-leucine Llysine Gmet,hionine L-ornithine L-phenylalanine r,-proline L-serine L-threonine L-tryptophane L-tyrosine Gvaline Ammonia * significant difference. a Concent,rat,ions expressed
Acids
and of Ammoniaa
1Iedium at beginning of run
MALARIAL
117
OOCYSTS
I at Beginning
A. eucalypti cells + Cuter tarsalis gut added to medium
of Run
and after
16-hr
A. eucalypti cells + C. tarsalis gut with Plasmodium relicturn oocysts added t,o medium
Incubation
Percent change in medium of Column 3 over Column 2 with estimated error of reading
2.32 0.70 2.42
3.90 1.36 4.08
2.10* 1.62 1.90*
-46 +19 -55
zt 5 It IO-20 It 3-5
1.88 0.94 Trace
3.46 1.60 0.20
2.98 1.02 Trace
-14 -36
f f
4.36 6.08 12.54 0.30 0.44 2.70 0.28 0.26 0.68 2.18 7.62 1.04 0.38 0.20 0.64 0.62
in mmoles/lOOO
7.34 See Gasparagine 10.38 20.98 0.48 0.74 4.56 0.44 0.38 1.10 3.84 12.98 1.76 0.64 0.32 1.12 I .26
4.58* and L-glutamine-not 6.70* 12.36* 0.30 0.46 2.76* 0.30 0.32 0.72 2.46* 8..58* 1.20 0.32 0.22 0.80 2.30*
l&20 lo-20 -
-38 separable -35 -41 - 37 -3s -39&5 -37 -16 -35 -36 -34 -32 -50 -Sl -39 +F;3
If 3-5 f xt f *
3-5 3-5 over 20 IO-20
3~ A * xt & f * * + zk
over 20 over 20 IO-20 5 3-B lo-20 IO-20 over 20 IO-20 5
ml of medium.
of incubation was that one contained an uninfected stomach of C. tarsalis and the other contained a C. tarsalis stomach bearing mature oocysts of Plasmodium relictum (Column 2 vs Column 3 in Table I). The stomachs used were heavily infected with oocysts; therefore, it is believed that the metabolism of the oocysts accounted for the marked reduction in the concentration of certain of the amino acids. It is recognized that the system is a complex one, and that the cells involved come from three different sources. However, simplification of the system has not been possible up to the present. Attempts to duplicate this finding with oocysts removed
from the stomachs and cultured in the medium with A. eucalypti cells were not successful since one could not dissect off enough oocysts to give reliable readings with the amino acid analyzer. It is possible that thin-layer chromatography of the medium before and after inoculation with individual oocysts might be sufficiently sensitive to measure their utilization of amino acids. ACKNOWLEDGMENTS
The authors wish to express their appreciation to Miss June Baumer for her skill in the use of the amino acid analyzer. The research was supported by Grant AI-00087 from NIAID, U.S.
118
BALLANDCHAO
Public Health Service, by Research Grant 254, Zoology, University of California, and by a Biomedical Sciences Support Grant to University of California. REFERENCES G. H., AND CHAO, J. 1971. The cultivation of Plasmodium relicturn in mosquito cell lines. Journal of Parasitology 57, 391395. GERARD, J. F., AND GILLES, R. 1972. The free amino-acid pool in Callinectes sapidus (Rathbun) tissues and its role in the osmotic intracelluIar regulation. Journal of Experimental Marine Biology and Ecology 10, 125-136. GRACE, T. D. C. 1962. Establishment of four strains of cells from insect tissues grown in BALL,
u&o. GRACE,
Nature
195,
788-789.
T. D. C. 1966. Establishment of a line of mosquito ( Aedes aegypti L. ) cells grown in vitro. Nature 211, 366-367. GRACE, T. D. C., AND BRZOSTOWSKI, H. W. 1966. Analysis of the amino acids and sugars in an insect cell culture medium during cell growth. Journal of Insect Physiology 12, 625-633. GREENE, A. E., CHARNEY, J., NICHOLS, W. W., AND CORIELL, L. L. 1972. Species identity of insect cell lines. In Vitro 7, 313-322. HAYASHI, Y., AND SOHI, S. S. 1970. Metabolism of Aedes aegypti cells grown in uitro. I. Incorpora-
tion of ‘H-uridine and %C-leucine. In Vitro 6, 146-152. Hsu, S. H., Lru, H. H., AND Surro~, E. C., JR. 1969. Further description of a subline of Grace’s mosquito ( Aedes aegypti L. ) cells adapted to hemolymph-free medium. Mosquito News 29, 439446. IGARASHI, F., SASAO, F., AND FUKAI, K. 1973. Effect of amino acids on growth of Singh’s Aedes albopictus cells and Japanese encephalitis virus. B&en Journal 16, 95-101. LANDUREAU, J. D., AND JOLLY, P. 1969. fitude des exigences dune lignee de cellules d’insectes (Souche EPa). Experimental Cell Research
54, 391-398.
S. S. 1969. Adaptation of an Aedes aegypti cell line to hemolymph-free culture medium. Canadian journal of Microbiology 15, 11971200. STANLEY, M. S. M. 1972. Cultivation of arthropod cells. In “Growth, Nutrition, and Metabolism of Cells in Culture” (G. H. Bothblat and V. J. Cristofalo, Eds.), VoF. II, pp. 327370. Academic Press, New York/London. YUNKER, C. E., VAUGHN, J. L., AND CORY, J. 1967. Adaptation of an insect cell line (Grace’s Antheraea cells) to medium free of insect hemolymph. Science 155, 1565-1566. SOHI,
39,
PARASITOLOGY
Use of Amino
Department
of
115-118
Acids
(19%)
by Plasmodium
relicturn
GORDON H. BALLANDJOWETT CHAO Biology, University of California, Los Angeles, (Accepted
for publication
May
Oocysts
California
in Vitro
90024
12, 1975)
BALL, G. H., AND CHAO, J. 1976. Use of amino acids by Plasmodium relicturn oocysts in vitro. Experimental Parasitology 39, 115-118. A comparison was made of the in vitro growth of the gut of Culex tarsalis in Grace’s insect culture medium, supplemented with fetal bovine serum in the presence of dividing cells of Antheraea eucalypti, with a similar preparation of a gut infected with oocysts of the avian parasite, Plasmodium relicturn. In the latter case, after 16 hr, significant decreases occurred in the concentration of arginine, asparagine, and glutamine combined, glutamic acid, glycine, histidine, lysine, proline, and serine. Lower and less marked decreased concentrations of alanine, p-alanine, cystine, isoleucine, leucine, methionine, ornithine, phenylalanine, threonine, tryptophane, tyrosine, and valine also took place. This indicated utilization of certain amino acids by the developing oocysts of P. relicturn in the presence of metabolizing insect cells. INDEX DESCRIPTORS: Plasmodium relicturn; Cubx tarsalis; Tissue culture; Amino acids.
INTRODUCTION
In the formulation of media for the cultivation of insect cells, the composition at first was largely empirical. Media proved successful for growing other tissues in vitro were employed, and it was found necessary to supplement these with insect hemolymph, usually of the same or of a related species (Grace 1962, 1966). Later it was shown that the hemolymph, often difficult to obtain or to keep unchanged, could be replaced by fetal bovine serum (Yunker, Vaughn, and Cory 1967, Hsu, Liu, and Suitor 1969, Sohi 1969). Grace and Brzostowski (1966) demonstrated that the cells of the moth, Antheraea eucalypti used 14 of 21 amino acids over 7 days culture while the concentration of seven others either increased or remained unchanged. Hayashi and Sohi (1970) showed that cells of Aedes aegypti ( Antheraea eucalypti?) incorporated leutine, the only amino acid tested, over a 5-hr period. Igarashi et al. (1973) found 115 Copyright All rights
1976 by Academic Press, Inc. o3 reproduction in any form reserved.
that “nonessential” amino acids stimulated growth over a 7-day period of Aedes albopictus cells. Landureau and Joll&s (1969) showed that 15 amino acids were required for optimum growth and transfer of cells of embryonic Periplaneta americana tissue followed for 7 days. Stanley (1972) summarized conditions for cultivating arthropod cells, including the conflicting results of various investigators on the utilization of amino acids. To the best of our knowledge, no such determination has been made for malarial oocysts developing in insect cell culture. MATERIALS
AND
METHODS
One Culex tars&s stomach, either noninfected or infected, was added in each experiment to 1 ml of medium, containing 1 X lo6 cells. The development of oocysts of PZasmodium relicturn attached to the midgut of Culex tarsalis and cultured in the presence of Grace’s insect cells in Grace’s medium
116
BALL
AND
containing fetal bovine serum has been described previously (Ball and Chao 1971). This medium was supplemented with 100 units of penicillin and 0.1 mg of streptomycin/ml. This cell line, originally believed to be that of Aedes aegypti, was found to be one of Antheraea eucalypti (Greene et al. 1972). Oocyst development was more favorable in this medium than in the medium without A. eucalypti cells or than in Singh’s medium containing cells of A. aegypti (Ball and Chao 1971). The amino acids in the culture medium were analyzed by a Beckman Model 120 amino acid analyzer. Samples were 50 ~1 in volume. Determinations were made of the medium alone at the start of the run, the medium containing an uninfected C. tarsalis stomach and A. eucalypti cells after 16 hr, and the medium containing a C. tarsalis stomach with attached mature oocysts of P. relictum and A. eucalypti cells after I6 hr. All tabulations shown are in millimoles/1000 ml of medium. Cultures were maintained at 22-23 C. The analysis of the medium that contained infected stomachs and A. eucalypti cells was the average of determinations of two parallel cultures. The inherent error of the analyzer decreased in significance as the differences in concentrations became greater. The reliability of the readings as determined by the operator were for differences of less than 0.2 m-moles, more than 20%; for 0.2-0.6 mmoles, *lo-20%; for 0.6-1.0 mmoles, *5-10%; for 1.0-2.0 mmoles, approximately *5%, and for more than 2.0 mmoles, 23-5s. A difference was not considered significant unless it was at least three times this inherent error. RESULTS
Over a 16-hr period, Grace’s medium containing growing cells of Antheraea eucalypti and a stomach of the mosquito Culex tarsalis showed an increase in the concentration of amino acids and of ammonia. On the other hand, if the mosquito
CHAO
stomach contained mature oocysts of Pla.smodium relicturn at the time it was added to the culture, the concentration of amino acids decreased over the same period of time in every case except for aspartic acid, which showed a small increase. The extent of decrease in amino acids of the infected stomach over the uninfected one in the presence of Antheraea eucalypti cells is indicated in Table I. Those amino acids which showed a significant decrease on the basis of our criteria in 16 hr of cultivation were arginine, asparagine, and glutamine (not separable), glutamic acid, glycine, histidine, lysine, proline, and serine. Lower but less marked decreases occurred in the concentrations of alanine, p-alanine, cystine, isoleucine, leucine, methionine, ornithine, phenylalanine, threonine, tryptophane, tyrosine, and valine. The increased concentration of aspartic acid was not significant. Ammonia, on the other hand, showed a significant increase. DISCUSSION
The increase shown in the concentration of amino acids over a I6-hr period in the media containing an uninfected stomach of Culex tarsalis and metabolizing cells of Antheraea eucalypti was probabIy due to the breakdown of some types of mosquito cells which did not survive in vitro. The method of amino acid assay documented in Table I did not indicate any transaminations or use of breakdown products by any amino acid. If these processes did occur, actual utilization of various amino acids by the oocysts would be greater than that recorded. Landureau and Jolles (1969) pointed out the evidence for transamination taking place in their cell cultures of Periplaneta americana. The large increase in the concentration of ammonia may indicate increased deamination activity at the level of various amino acids (Gerard and Gilles 1972). The only difference between the two media assayed for amino acids after 16 hr
AMINO
ACID
METABOLISM
IN
TABLE Concentration Amino
of Amino
acids
Larginine baspartic acid basparagine, L-glutamine (not separable) Lalanine p-&nine 4 cystine L-glut,amic acid 1,glutamine Glgcine I.-hi&dine r,isoleucine I,-leucine Llysine Gmet,hionine L-ornithine L-phenylalanine r,-proline L-serine L-threonine L-tryptophane L-tyrosine Gvaline Ammonia * significant difference. a Concent,rat,ions expressed
Acids
and of Ammoniaa
1Iedium at beginning of run
MALARIAL
117
OOCYSTS
I at Beginning
A. eucalypti cells + Cuter tarsalis gut added to medium
of Run
and after
16-hr
A. eucalypti cells + C. tarsalis gut with Plasmodium relicturn oocysts added t,o medium
Incubation
Percent change in medium of Column 3 over Column 2 with estimated error of reading
2.32 0.70 2.42
3.90 1.36 4.08
2.10* 1.62 1.90*
-46 +19 -55
zt 5 It IO-20 It 3-5
1.88 0.94 Trace
3.46 1.60 0.20
2.98 1.02 Trace
-14 -36
f f
4.36 6.08 12.54 0.30 0.44 2.70 0.28 0.26 0.68 2.18 7.62 1.04 0.38 0.20 0.64 0.62
in mmoles/lOOO
7.34 See Gasparagine 10.38 20.98 0.48 0.74 4.56 0.44 0.38 1.10 3.84 12.98 1.76 0.64 0.32 1.12 I .26
4.58* and L-glutamine-not 6.70* 12.36* 0.30 0.46 2.76* 0.30 0.32 0.72 2.46* 8..58* 1.20 0.32 0.22 0.80 2.30*
l&20 lo-20 -
-38 separable -35 -41 - 37 -3s -39&5 -37 -16 -35 -36 -34 -32 -50 -Sl -39 +F;3
If 3-5 f xt f *
3-5 3-5 over 20 IO-20
3~ A * xt & f * * + zk
over 20 over 20 IO-20 5 3-B lo-20 IO-20 over 20 IO-20 5
ml of medium.
of incubation was that one contained an uninfected stomach of C. tarsalis and the other contained a C. tarsalis stomach bearing mature oocysts of Plasmodium relictum (Column 2 vs Column 3 in Table I). The stomachs used were heavily infected with oocysts; therefore, it is believed that the metabolism of the oocysts accounted for the marked reduction in the concentration of certain of the amino acids. It is recognized that the system is a complex one, and that the cells involved come from three different sources. However, simplification of the system has not been possible up to the present. Attempts to duplicate this finding with oocysts removed
from the stomachs and cultured in the medium with A. eucalypti cells were not successful since one could not dissect off enough oocysts to give reliable readings with the amino acid analyzer. It is possible that thin-layer chromatography of the medium before and after inoculation with individual oocysts might be sufficiently sensitive to measure their utilization of amino acids. ACKNOWLEDGMENTS
The authors wish to express their appreciation to Miss June Baumer for her skill in the use of the amino acid analyzer. The research was supported by Grant AI-00087 from NIAID, U.S.
118
BALLANDCHAO
Public Health Service, by Research Grant 254, Zoology, University of California, and by a Biomedical Sciences Support Grant to University of California. REFERENCES G. H., AND CHAO, J. 1971. The cultivation of Plasmodium relicturn in mosquito cell lines. Journal of Parasitology 57, 391395. GERARD, J. F., AND GILLES, R. 1972. The free amino-acid pool in Callinectes sapidus (Rathbun) tissues and its role in the osmotic intracelluIar regulation. Journal of Experimental Marine Biology and Ecology 10, 125-136. GRACE, T. D. C. 1962. Establishment of four strains of cells from insect tissues grown in BALL,
u&o. GRACE,
Nature
195,
788-789.
T. D. C. 1966. Establishment of a line of mosquito ( Aedes aegypti L. ) cells grown in vitro. Nature 211, 366-367. GRACE, T. D. C., AND BRZOSTOWSKI, H. W. 1966. Analysis of the amino acids and sugars in an insect cell culture medium during cell growth. Journal of Insect Physiology 12, 625-633. GREENE, A. E., CHARNEY, J., NICHOLS, W. W., AND CORIELL, L. L. 1972. Species identity of insect cell lines. In Vitro 7, 313-322. HAYASHI, Y., AND SOHI, S. S. 1970. Metabolism of Aedes aegypti cells grown in uitro. I. Incorpora-
tion of ‘H-uridine and %C-leucine. In Vitro 6, 146-152. Hsu, S. H., Lru, H. H., AND Surro~, E. C., JR. 1969. Further description of a subline of Grace’s mosquito ( Aedes aegypti L. ) cells adapted to hemolymph-free medium. Mosquito News 29, 439446. IGARASHI, F., SASAO, F., AND FUKAI, K. 1973. Effect of amino acids on growth of Singh’s Aedes albopictus cells and Japanese encephalitis virus. B&en Journal 16, 95-101. LANDUREAU, J. D., AND JOLLY, P. 1969. fitude des exigences dune lignee de cellules d’insectes (Souche EPa). Experimental Cell Research
54, 391-398.
S. S. 1969. Adaptation of an Aedes aegypti cell line to hemolymph-free culture medium. Canadian journal of Microbiology 15, 11971200. STANLEY, M. S. M. 1972. Cultivation of arthropod cells. In “Growth, Nutrition, and Metabolism of Cells in Culture” (G. H. Bothblat and V. J. Cristofalo, Eds.), VoF. II, pp. 327370. Academic Press, New York/London. YUNKER, C. E., VAUGHN, J. L., AND CORY, J. 1967. Adaptation of an insect cell line (Grace’s Antheraea cells) to medium free of insect hemolymph. Science 155, 1565-1566. SOHI,
