J. PROTOZOOL. 2 4 ( 3 ) , 437-441 (1977)

Cultivation of Leishmania donovuni and Leishmania braziliensis in Defined Media: Nutritional Requirements ROLF F. STEIGER*t and ERICA STEIGER Laboratory of Parasatology, The Rockefeller University, New York, New York 10021$

SYNOPSIS. Nutritional requirements of promastigotes of Leishmania donouani and Leishmania braziliensis were studied in modifications of a simple defined culture medium. “Continuous growth,” considered as propagation through 10 successive passages, was supported by inorganic salts, 14 L-amino acids (arginine, cysteine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, valine) , glucose, adenosine, and a mixture of 11 vitamins and related growth factors. Purified defatted bovine serum albumin proved beneficial. The nutritional needs of the abote species of Leishmania differ from those of 2 other hemoflagellate species, Leishmania tarentolae and Crithidia fasciculnta. for which glucose, proline and glutamine were found to be nonessential. I t is suggested that lower hemoflagellates may be capable of synthesizing these substrates de novo. Leishmania donovani and L. braziliensis required higher lwels of folk acid than L. tarentolae, probably due to the fact that folates are involved as cofactors in the biosyntheses of pyrimidines and serine. Although the mixtures reported here cannot be regarded as “minimal essential” media, they are considerably less complex than the ones employed so far for cultivating hemoflagellates, and are therefore well suited for studies related to nutrition and biosynthctic capabilities of Trypanosomatids.

Index Key Words: Leishmania donovani; Leishmania braziliensis; promastigotes; defined culture media; nutrition.

HEMICALLY defined media are available for the cultivation of a few hemoflagellate species (for review see Ref. 44), which, in the genus Leishmania, include L. tarentolae

C

(Wenyon) ( 4 2 ) , a parasite from lizards, as well as 2 human pathogens, L. donouani (Laveran & Mesnil) and L. braziliensis (Vienna) ( 3 9 ) . Growth under defined conditions permitted extensive work on the nutritional requirements of Crithidia fasciculata (Ltger) (20) and L. tarentolae (42, 43). For the latter species the central role of proline as an energy source and a key intermediate in amino acid biosynthesis was demonstrated ( 2 4 ) . In the present report we describe nutritional experiments with promastigotes of L. donouani and L. braziliensis cultivated in deficient versions of the defined medium RE I (39). Special emphasis is placed on glucose and amino acid requirements as compared to those of related parasites. MATERIALS AND METHODS Parasites.-Leishmania donouani, a cloned derivative ( 15) of Sudan strain IS, was maintained in hamsters, and amastigotes harvested as described elsewhere ( 1 9 ) , except for using culture medium (see below) in the tissue homogenization step. A Leishmania braziliensis-like strain (Hopkins) was obtained from Dr. J. Keithly in its first subculture in Tobie’s medium (41). Amastigotes of this strain had been isolated in 1971 from a cutaneous lesion of a patient returning from the southeastern regions of Venezuela, and produced mucocutaneous nasal lesions in hamsters. Biopsy material from a hamster nose was kept as frozen stabilates until early 1976, when some of the thawed parasites were inoculated into Tobie blood-agar slants. Preparatory Cultivation.-Cultures in medium RE I (39) supplemented with 10% (v/v) fetal bovine serum (FBS; Microbiological Associates), were initiated with amastigotes of L. donouani (final concentration 107 cells/ml) and promastigotes of L. braziliensis (final concentration 2 X lo7 cclls/ml) derived

* Recipient of a Fellowship from the Swiss National Foundation for Scientific Research, Nr. 831.372.75, with additional support from the Bade Foundation for Experimental Zoology. 7 Present address: International Institute of Cellular and Molecular Pathology (ICP), Avenue Hippocrate 75, B-1200 Bruxelles, Belgique. $Address to which reprint requests from the U.S.A. should be sent.

from Tobie’s culture. Promastigotes of both specks were subsequently grown in duplicate disposable sterile plastic screw-cap tubes (Falcon, Nr. 2025) a t 27 _C 1 C, and subcultured in the late log phase (-2 x 107 cells/ml) at 4-day intervals. The inoculum was usually adjusted to 106 cells/rnl in a final volume of 2 ml of medium/culture tube. Log-phase parasites from the 4th subpassage were then washed 3X by centrifugation (1,500 g ) , resuspended in serum-free R E I11 (Table 2 ) , and similarly subcultured 6X in the latter medium. Cells of the 7th subpassage ( C 7 ) in RE 111 were used to seed the experimental media described below, or were frozen in R E I11 10% (v/v) glycerol and stored at -70 C until required for replicate experiments. Nutritional Experiments.-For studying glucose and amino acid requirements of L. donouani and L. braziliensis, REN medium (Table 1 ) was devised, which lacked glucose and contained only the 10 “essential” L-amino acids (arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine). As summarized in Table 2, 19 diffrrent modifications of REN were then prepared by adding glucose and/or further L-amino acids (cysteine, cystine, glutamic acid, glutamine, proline, serine, tyrosine) from freshly prepared 1 0 0 ~ concentrated aqueous stock solutions ( filter-sterilized through 0.22 p m Millex disposable filter units, Millipore) at the same final concentrations as in RE I. Requirements for other growth factors, i.e. “essential” amino acids, vitamins, hemin, purines, albumin, were examined in RE I and RE 111 media by deleting single substrates or by reducing their concentrations 2-8-fold. Parasite growth in the aforementioned riicdia was followed after inoculating 2 x lo6 C7 cells in 0.1 ml into 1.9 nil of medium; transfers to fresh medium were made every 4 days using the same inoculum. All nutritional experinicnts with L. donouani and L. braziliensis were repeated once within a 10-week period. Growth Parameters.-“Continuous growth” \;as considered propagation of promastigotes over 10 successive passages. Daily samples (0.1 ml aliquots) were aseptically withdrawn in each subpassage from 2 experimental culture tubes, then fixed and diluted 50X with cold 1% (w/v) formalin-PHS for counting. Cell counts were made in Petroff-Hausser bacteria counters. Average counts of organisms from the duplicate tubes were plotted vs time on semilog paper; generation times ( G ) were determined from the linear portions of the curves (log phase),

437

+

438

NUTRITION OF Leishmania TABLE 1. Composition

A)t

B)t

C) D)

NaCl 8000 KCI 400 MgSOd. 7H20 200 Na2HPOa.2H20 60 KH2POh 60 CaCI, 70 L-Arginine HC1 200 L-Histidine 100 L-Isoleucine 100 L-Leucine 300 L-Lysine HCI 250 L-Methionine 50 L-Phenylalanine 100 L-Threonine 400 1.-Tryptophan 50 L-Valine 100 NaHCOs 1000 HEPES (60 r n ~ )14250 Adenosine 20

of

E)t

F)? G)

medium REN.* D-Biotin 1 Choline CI 1 Folic acid: 10 i-Inositol 2 Niacinamide 1 D-Ca Pantothenate 1 Pyridoxal HCl 1 Riboflavin 0.1 Thiamine HCI 1 Lipoic acid 0.4 Bovine Albumins (defatted) 15 Hemin: 5 Phenol red 5 Redist. HZO Q.S. 1000 ml pH adjusted with 1 N NaOH to 7.3-7.4 Tonicity I 402 mOsm

* .411 quantities = mg, liter. t A.B,E, from frozen stock solutions:

2 x , 5>< and 1 0 0 ~(BME Vitamins, GIBCO), respectively. Lipoic acid ( F ) dissolved and added in 50 p1 EtOH. $ Hemin and additional folic acid ( 9 mgjliter) added from separate solutions freshly prepared with 1 s h’aOH. I Prepared as described in Materials and Methods and their nican values and standard errors (Table 2 ) computed from the first and all subsrqucnt passages. Preparation of Culture Media.-The media n’ere prepared as outlined previously (39) and in Table 1; they were filtersterilized by pressurc through Millipore HAWP 04-700 (0.35 p m ) , then stored in the dark at 4 C. The same batch of R E N was used throughout this study within 10 wwks after preparation. Bovine albumin \vas drfatted ( 8 ) , and batches prepared as described previously ( 10 ; . Concmtrations of defatted bovine albumin \\‘ere chrckcd at 279 nni ( E l & = 6.67) in a Beckman DU-2400 Spectrophotorn(,ter. Osmolality lvas measured by freez-

TABLE 2. A niino acid requirements

of

ing-point depression in an automated osmometer (Typc 3D, Advanced Instruments, Inc.). Chemicals.-Reagents of the purest grades available were purchased from J. T. Baker Co. (D-glucose, inorganic salts), Schwarz/Mann ( L-amino acids), and Sigma Chemical Co. ( Lamino acids, HEPES, purines, hemin, folic acid, lipoic acid). Phenol red (0.5%) was from Grand Island Biological Co. ( G I B C O ) , and bovine serum albumin (99% pure, crystallized) from Miles Research Products, Elkhart, Indiana. RESULTS Idrntical results were obtained with both species of Leishinania. Amino Acid and Glucose Requirements (Table 2 ) .--REN did not support growth of promastigotes; the cells multiplied only moderately in their first subpassage, with a G exceeding 48 hr. Inclusion of 2.6 mM proIine and/or 11 mM glucose was somewhat beneficial, since the parasites could be subcultured 2 and 3 times, respectively, before they died off. T h e G values were smaller, ranging from 40.5-43.0 hr. Further addition of 2.05 mM glutamine did not stimulate multiplication significantly; however, 0.28 r n A 4 tyrosine in addition to the above 3 substrates led to 4 continuous subcultures with a clearly shorter G of 38.2 hr. Subsequent enrichment of the medium with 0.32 mM cysteineHCI allowed continuous growth of the parasites with a mean G of 17.3 hr at 27 1 C. This experimental medium is henceforth referred to as R E 111. Growth of both species of Leishmania in this medium is depicted in Fig. 1. When glutamine was rcplaced by 2 mar glutamic acid, the cells stopped growing beyond thc 5th subpassage. As can be expected, the more complex medium RE I, which contains a total of 1 7 amino acids, i x . cystine, glutamic acid and serine in addition to the 14 present in R E 111, supported continuous growth of the parasites equally well, although with

*

Leishmania donovani and L. braziliensis pronastigotes.

_ _ _ _ _ ~

_.

Y2,Cys

Cvs

-

-

-

-

++ + ++ ++ ++ -

-

-

-

-

-

-

+++

Amino acids added* Gln Glu Pro -

-

++ ++ +++ +

-

++ ++

-

-

-

-

+ -

-

+++

-

++ ++ +++ ++ +-

++

Ser

Tyr

-

-

-

-

-

-

-

++ +

-

-

-

+++ ++ +

++ ++ ++

Maximal number of continuous subcultures

Glucose*

+- (=REN) ++-

1 3 2 3 1 3

+-

2 2 2

++ (=RE ++ + ++ + (=RE + -

4

2 111)

I)

>10

5 2 2 2 3 >I0 6 5

Population doubling time ( h r ) I’ >48 40.5 & 3.9 43.0 2 6.1 41.5 2 6.8 >48 42.4 f 7.1 42.8 & 7.2 42.3 2 5.9 43.0 rt 6.5 38.2 2 4.7 42.0 & 4.3 17.3 ? 2.4. 25.6 2 3.9 43.0 f 5.0 40.2 2 6.1 40.9 2 3.2 41.8 ? 4.2 21.0 2.1 28.5 i 3.7 31.2 2 4.8

*

~ _ glucose added to REX from fresh sterile stock solutions ( l o o > < ); final concentrations (mg/liter) = glucose, 2000; cysteine, cystine and tyrosine, 50; glutamic acid, glutamine and proline, 300; serine, 200. Tonicity in all experiments adjusted with NaCI to 120 mOsm. t Means 2 S.E. of at least 4 determinations. The values listed are those of L. donouani; data from L. braziliensis are not significantly different (t-test: P > 0.1).

_~______

* Individual L-amino acids and

_

439

NUTRITION OF Leishmania DISCUSSION A

Fig. 1. Growth of Leishmania donovani )-.-( and L. bratiliensis (- - -0- - -) promastigote in defined medium RE 111. Counts represent means -1- SD’s of 9 determinations (triplicate cultures over 3 passages).

a somewhat longer G, which equals 21 hr. By t-test, this difference in G values was found to be not significant ( P > 0.05). Cultivability of the parasites was no longer possible after either glucose or proline was deleted from both media. Likewise, oniission of glucose and/or proline from an enriched medium, containing alanine, aspartic acid, glutamic acid, and glycine in addition to the above 17 amino acids of RE I, resulted in growth inhibition. Continuous growth was impossible also when any one of the 10 “essential” L-amino acids (arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine) was deleted from the medium. Omission of isoleucine and leucine in combination with hydroxyurea treatment (0.2 mg/liter), has proved promising in preliminary synchronization attempts (unpublished observation) as monitored by thymidine incorporation and cell counts. Other Growth Factors.-Reduction of bovine albumin concentration in R E I and R E I11 (7.5, 3.75, 1.875 mg/liter) correlated with prolongation of mean G (25.2, 31.0, and 38.2 hr, respectively). When albumin was lacking, G equaled -40 hr, although in all cases cell growth through more than 9 subpassages has been achieved. Concentrations of hemin and folic acid should be lowered to 1.25 mg and 2.5 mg/liter, respectively, without an adverse effect on the growth pattern. Below these levels, however, cell viability was clcarly reduced and cultivation inconsistent. Addition of adenosine at 10 mg/liter did not retard growth, but at half this concentration growth was slower. This also applied to guanosine when substituted for adenosine. Defined medium R E I, used originally in successful cultivation of L. donouani and L. braziliensis, also contained, apart from 3 additional amino acids, sodium acetate, guanosine as well as adenosine, menadione (vitamin K3), vitamine A (trans-retinoic acid), ascorbic acid and vitamin El, (cyanocobalamin) Therefore, delction of these reagents in the preparation of the simpler formula R E 111 did not adversely affect parasite growth. Further Experiments.-We were unable to grow promastigotes of either species in the considerably more complex defined Trypanosoma brucei media HX 25 and HX 28 (10, 1 2 ) , in defined medium C ( 4 2 ) , and in a defined insect trypanosomatid medium ( 3 5 ) .

.

Continuous cultivation of Leishmania donouani and L. braziliensis promastigotes requires inorganic salts, D-glUcOSC and the following 14 L-amino acids : arginine, cysteine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalaninr, proline, threonine, tryptophan, tyrosine and valine. In addition, adenosine or guanosine, as well as many vitamins and related growth factors, including folk acid and hemin, are essential. Glucose and amino acid requirements of the aforementionrd Leishmania spp. differ significantly from those of othcr henioflagellates (for review see Ref. 44). Glucosc is required for amastigote-promastigote transformation of L. donoz’ani ( 3 6 ) , but it is dispensable for the growth of lower Trypanosomatidae provided certain glycogenic amino acids are supplemcntcd, such as proline and glutamic acid in the case of L. tarentolae ( 2 4 ) . Although glucose strongly stimulates oxygen consumption of the above species ( 2 3 ) and L. enrietti ( 2 7 ) , it does not appear to be an essential building block for biosynthetic pathways of these organisms; in “lower” hemoflagellates gluconeogenesis may therefore play an important role. T h e initial enzymic stcps necessary for the gluconeogenic schemc of substrate utilization, with pyruvate as the key intermediate, have been demonstrated ( 1, 21). I n trypanosomatids, which depend on glycolysis, cfficient carrier-mediated hexose transport systems are present ( 2, 37). Moreover, in defined or semidefined media glucose is consumed at high rates by T . brucei ( l l ) , L. donouani and L. braziliensis (40) throughout the growth cycle. In more complcx media, utilization of glucose has been described as restricted to late log and stationary growth phases ( 3 0 ) ; delaycd utilization of exogenous glucose under nondefincd conditions may reflect availability of nutrients that have to be synthesized de nouo in defined media. Leishmania spp. have the tricarboxylic acid cycle, and their glycolysis proceeds via the Embden-Meyerhof pathway and the pentose phosphate shunt (28). Reoxidation of NADH is probably mitochondria1 ( 22), and the 1.-a-glyccrophosphate dehydrogenase/oxidase system characteristic of T . brucei ( 9 ) seems to be absent, as evidenced by respiration insensitive to salicylhydroxamic acid (unpublished observation). The role of proline as a major energy substrate has bceri demonstrated in promastigotes of L. donouani (25) and L. tarentolae (23) as well as in procyclics of T . rhodesicnsc ( 5 ) . I t was shown ( 25) that alanine, arginine, cystine, glutamate, glycine, histidine, isoleucine, leucine, lysine and threoninc are produced from 14C-labeled glucose and proline. Seven of these amino acids are required by L. donouani and L. brariliensis undcr the conditions described elsewhere (39) and in the present paper, w e n when all other amino acids are available to the cells. This probably means that the 7 “essential” amino acids are not synthesized in amounts sufficient for continuous growth. Proline, unlike this amino acid in L. donouani, is dispensable for L. tarentolae provided glutamate, methionine, isoleucine, alanine and aspartate are added to the 10 basal amino acids ( 2 4 ) . Therr is evidence that proline can be formed de n o w from arginine and ornithine in the latter species (45). I t is surprising that both sulfur-containing amino acids, cystcinc and methionine, are needed in our media, since either of them can serve as an organic sulfur in L. tarentolae ( 1 4 ) . Methionine may play a key role in fatty acid methylation ( 2 9 ) . Leishmania cannot dispense with threonine, which may be a precursor of acetate production and fatty acid biosynthesis, much as in T . brucei procyclics ( 12). Glutamic acid is not essential for all hemoflagellates studied, probably because it is readily produced by

440

NuTRrrIox OF Leishmania

transamination. Glutamine, on the other hand, cannot be omitted or replaced by glutamate in our media, which implies that the “lower” trypanosomatids must be able to synthesize the former amino acid d e nouo. Since the amino acid balance of our media has not been altered quantitatively in the course of this study, R E I11 cannot be regarded as a “minimal cssential“ medium Lvith regard to amino acids. Likewise, the minimal requirements of L. donocani and L. braziliensis for vitamins and related growth fatcors have not fully been established. Folic acid had to be supplied a t a n unusually high concentration ( 2.5 nigjliter) probably to meet demands for unconjugated pteridines, which are cofactors in amino acid hydroxylation ( 4 ); folates are indispensable for anabolic reactions involving onc-carbon transfers, such as biosyntheses of serine and pyrimidines (thymidylate) ( 1 7 ) . In contrast with L. donovani and L. brazilicnsis, growth of L. tarentolae is not affected when the folic acid concentration is reduced to 1.7 pg/liter (43). This quantitative difference may be based on the fact that pyrimidines are already included in Trager’s Medium C, used for the nutritional experiments with the latter species (42j. Only one purine, adenosine or guanosine, is required in our experimental series, ivhich suggests that Leishmania depends on the same purine salvage pathway as T r y p a n o soma (18). T h e hemin requirement is a general feature of hemoflagellates ( 4 4 ); exogenous hemin can be replaced by catalase ( 1 6 ) in L. tarentolae, and is not required by hemoflagellates containing synibiotrs capable of synthesizing heniin d e noco ( 7 ) . The beneficial effect of albumin on the growth of hemoflagellates, although at a much higher concentration, has been observed before with T . brucei ( 10). Albumin may function ( a ) as a detoxifying agent by trapping surface active fatty acids ( 1 3 ) ; ( b ) as a carrier of heniin (31) and metals ( 3 4 ) ; and ( c ) as a stimulant of pinocytosis and therefore as a nutritional factor. Since both L. donovani and L. brazilicnsis grow in media containing Na+, K+, Ca+, Mg”, Cl-, PO,3- and CO,”, without additional trace metals, it can be concluded that the latter are supplied as reagent contaminants. Likewise, artificial chelators (e.g. E D T A ) are not required; apart from excreted organic acids (citrate, fumarate], aniino acids may function as metal binders (compiled in 33 j . I t is impossible, a t this point, to correlate the nutritional requirements of the 2 mammalian leishmanias studied lvith the conditions prevailing inside the vector, since the biochcmistry and physiology of Phlebotomid sandf1ic.s are virtually unknoivn ( 2 6 ) . Dependence on proline by Leishmania promastigotes may reflect the key role of this amino acid for energy production in Phlebotomus, as has been demonstrated before in Glossina ( 6 ) . In the present study we describe the continuous cultivation of two human leishmanias in defined medium RE 111, a simplified version of medium R E I ( 3 9 ) . \Vhen supplemented Lvith 2.5-10k (v/v) dialyzed or regular FBS, hoth media support direct transformation and excellent growth of T . brucai ( 3 8 ) and L. donouani. Compared with other defined (10, 1 2 ) or semidefined (3, 32) media usrd for the cultivation of pathogenic hemoflagellates, media of the RE series are considerably lesy complex, and may therefore be the mixtures of choice for studies dealing with the ccli biology, hiorhemistry, nutrition, and chemotherapy of pathogcnic species of L f i s h m a n i a .

ACKNO\\’LEDGEhlEIYTS We tvish to thank Prof. 14’. Tragvr for very helpful advice, Dr.

D. M. Dtvyer for \.aluahle suggestions, Dr. K.-P. Charig for

collaboration in the maintenance of the parasites, Mrs. D. Sutter ( T h e S e w York Hospital) for the osmometric measurements, and Mrs. D. Greene for help in the preparation of this manuscript. REFERENCES 1. Bacchi CJ, Ciaccio EI, Kaback DB, Hutner SH. 1970. Oxaloacetate production via carboxylation in Crithidia fasciculata preparations. J . Protozool. 17, 305-11. 2. Balber AE, Patton CL. 1977. Trypanosoma brucei: glucose transport and glycolytic regulation. J . Protozool., in press. 3. Berens RL, Brun R, Krassner SM. 1976. A simple monoplasic medium for axenic culture of hemoflagellates. ]. Parasitol. 62. 360-5. 4. Blakley RL. 1969. T h e Biochemistry of Folic Acid and Related Pteridines. North-Holland, Amsterdam, pp. 332-62. 5. Bowman IBR, Srivastava HK, Flynn IW. 1972. Adaptations in oxidative metabolism during the transformation of Trypanosoma rhodesiense from bloodstream into culture form, in Van den Bossche J, ed., Comparative Biochemistry of P.arasites, .4cademic Press, New York, pp. 329-42. 6. Bursell E, Slack E. 1976. Oxidation of proline by sarcosomes of the tsetse fly, Glossina morsitans. Insect Biochem. 6, 159-67. 7. Chang KP, Trager W. 1974. Nutritional significance of symbiotic bacteria in two species of hemoflagellates. Science 183, 531-2. 8. Chen RF. 1967. Removal of fatty acids from serum albumin by charcoal treatment. J . Biol. C h e m . 242, 173-81. 9. Clarkson AB Jr, Brohn FH. 1976. Trypanosomiasis: An approach to chemotherapy by the inhibition of carbohydrate catabolism. Science 194, 204-6. 10. Cross GAM, Manning JC. 1973. Cultivation of Trypanosoma brucei sspp. in semi-defined and defined media. Parasitology 67, 3 15-31. 11. . Klein RA, Baker JR. 1975. Trypanosoma cruzi: growth, amino acid utilization and drug action in a defined medium. A n n . T r o p . M e d . Parasitol. 69, 513-4. , Linstead DJ. 1975. Utilization of amino 12. -,acids by Trypanosoma brucei in culture: L-threonine as a precursor for acetate. Parasitology 71, 31 1-26. 13. Cunningham LV, Kazan BH, Kuwahara SS. 1972. Effect of long-chain fatty acids on some Trypanosomatid flagellates. J . Gen. Microbiol. 70, 491-6. 14. DaCruz FS, Krassner SM. 1971. Assimilatory sulfate reduction by the hemoflagellate Leishmania tarentolae. J. Protorool. 18, 718-22. 15. Dwyer, DM. 1976. Leishmania donovani: surface membrane carbohydrates of promastigotes. Ex@. Parasitol., in press. 16. Gaughan PLZ, Krassner SM. 1971. Hemin deprivation in culture stages of the hemoflagellate Leishmania tarentolae. C o m p . Biochem. Physiol. 39B, 5-18. 17. Herbert V, Das KC. 1976. The role of Vitamin B, and folk acid in hemato- and other cell-poiesis, i n Munsori PL, Diczfalusy E, Glover J, Olson RE, eds., Vitamins and HormonesAdcances i n Research and Applications, Academic Press, New York 34, 1-30. 18. J.affe JJ, Gutteridge WE. 1973. Purine and pyrimidine metabolism in protozoa, in de Puytorac P, Grain J, eds., Actualitts Protoroologiques, RCsumk des Discussions des Tables Rondes du 4” C0ngri.s International de Protozoologie, 2-9 Septembre 1973, Ciermont-Ferrand, 1, 23-35. 19. Keithly JS. 1976. Infectivity of Leishmania donovani amastigotes and promastigotes for golden hamsters. J . Prototool. 23, 244-5. 20. Kidder GW, Dutta BM. 1958. The growth and nutrition of Crithidia fasciculata. J . Gen. Microbiol. 18, 62 1-38. 21. Klein RA, Linstead DJ, Wheeler MV. 1975. Carbon dioxide fixation in trypanosomatids. Parasitology 71,93-107. 22. Krassner SM. 1966. Cytochromes, lactic dehydrogenase and transformation in Leishmania. J . Protozool. 13, 286-90. 23. 1969. Proline metabolism in Leishmania tarentolae. E x p . Parasitol. 24, 348-63. 24. __ , Flory B. 1971. Essential amino acids in the culture of Leishmania tarentolae. J . Parasitol. 57, 91 7-20. _ _ 1972. Proline metabolism in Leishmania 25. -. donocani. J. Protozool. 19, 682-5. ~

~

441

NUTRITION OF Leishmania 26. Lewis DJ. 1971. Phlebotomid Sandflies. Bull. W l d . Hlth. Org. 44, 535-51. 27. Mancilla R, Naguira C, Lanas C. 1965. Metabolism of glucose labeled with carbon-14 in Leishmania enrietti. Nature, Lond. 206, 27-8. 28. Martin E, Simon MW, Schaefer FW 111, Mukkada AJ. 1976. Enzymes of carbohydrate metabolism in four human species of Leishmania: a comparative study. J. Prototool. 23, 600-7. 29. Meyer H, Holz GG. 1966. Biosynthesis of lipids by kinetoplastid flagellates. J. Biol. Chem. 241, 5000-7. 30. Mukkada AJ, Schaefer FW 111, Simon MW, Neu C. 1974. Delayed in vitro utilization of glucose by Leishmania tropica promastigotes. J . Prototool. 21, 393-7. 31. Muller-Eberhard U, Morgan WT. 1975. Porphyrinbinding proteins in serum. A n n . N . Y . Acad. Sci. 244,624-49. 32. O’Daly JA. 1975. A new liquid medium for Trypanosoma (Schirotrypanum) crutzi. J . Protorool. 22, 265-70. 33. O’Sullivan WJ. 1969. Stability constants of metal complexes, in Dawson RMC, Elliott DC, Elliott WH, Jones KM, eds., Data for Biochemical Research, 2nd ed., Academic Press, New York 1, 131-81. 34. Peters T Jr. 1975. Serum Albumin, in Putnam FW, ed., T h e Plasma Proteins-Structure, Function and Genetic Control, 2nd ed., Academic Press, New York, 1, 131-81. 35. Roitman C, Roitman I, De Azevedo HP. 1972. Growth of an insect trypanosomatid a t 37 C in a defined medium. J. Prototool. 19, 346-9. 36. Simpson L. 1968. The leishmania-leptomonad trans-

formation of Leishmania donouani: nutritional requirements, respiration changes and antigenic changes. J. Protozool. 15, 201-7. 37. Southworth GC, Read CP. 1970. Specificity of sugar transport in Trypanosoma gambiense. J. Protorool. 17, 396-9. 38. Steiger RF. 1976. A new culture medium for Trypanosoma brucei. J. Protorool. 23, 33A-4A. 39. __ , Steiger E. 1976. A defined medium for cultivating Leishmania donouani and L. brasiliensis. J. Parasitol. 62, 1010-11. 40. --, Meshnick SR. 1977. Amino acid and glucose utilization of Leishmania donouani and L . braziliensis. Trans. R. Soc. T r o p . M e d . Hyg.,in press. 41, Tobie EJ, von Brand T, Mehlman B. 1950. Cultural and physiological observations on Trypanosoma rhodesiense and Trypanosoma gambiense. J . Paratitol. 36, 48-54. 42. Trager W. 1957. Nutrition of a hemoflagellate (Leishmania tarentolae) having an interchangeable requirement for choline or pyridoxal. J. Protozool. 4, 269-76. 43. __ 1969. Pteridine requirement of the hemoflagellate Leishmania tarentolae. J. Protozool. 16, 372-5. 44. _ _ 1974. Nutrition and biosynthetic capabilities of flagellates : problems of in vitro cultivation and differentiation, in Trypanosomiasis and Leishmaniasis with Special Reference t o Chagas’ disease, CIBA Foundation Symposium 20 (new series), Elsevier-Excerpta Medica, North-Holland, Amsterdam, London, New York, pp. 225-45. 45. Wagner KP, Krassner SM. 1976. Leishmania tarentolae: proline anabolism in promastigotes. Exp. Parasitol. 39, 186-94.

J. PROTOZOOL. 24(3), 441-444 (1977).

Regulation by Ammonium of Variation in Heterotrophic CO, Fixation by Euglena gracilis During Growth Cycles* J. G . PEAK and M. J. PEAK Department of Zoology and Entomology, Rhodes University, Grahamstown, South Africa

SYNOPSIS. Heterotrophic (dark) COZ fixation by Euglena gracilis strain Z varies with phase of batch culture growth and mode of nutrition. Increases in the fixation during growth cycles correlate closely with the depletion of exogenous NH4+from the medium during growth. I t is demonstrated that exogenous NH4’ regulates a component of heterotrophic COe fixation and that another component is independent of NHa+. This is true for cells grown heterotrophically (glucose, dark), autotrophically (CO2, light) and for a permanently bleached strain ( E . gracilis SB3). Some kinetics of the NH,’ regulation are presented.

Index Key Words: Euglena gracilis; heterotrophic COz fixation ; ammonium. EVEDAHL ( 5 ) has reviewed the available literature on heterotrophic (dark) CO, fixation by Euglena. The nature of the fixation is still unknown; however, the range of compounds fixed may be unique ( 6 ) . Dark C 0 2 fixation in Euglena may be involved in anaplerotic replenishment of the tricarboxylic acid cycle compounds, drained by biosyntheses during growth ( 7 ) . Large transient elevations in dark fixation occur a t certain stages of batch growth cycles when Euglena are grown either photoautotrophically or heterotrophically on glucose but not when grown heterotrophically on the 2-C substrates, acetate or ethanol ( 7 ) . These results suggest that induction of glyoxylate cycle metabolism spares a requirement for dark CO, fixation. Since the glyoxylate cycle obviates a need for anaplerotic replenishment of tricarboxylic cycle intermediates, it may be inferred that dark CO, fixation is normally involved in this replenishment.

* This investigation was supported by grants from the Council for Scientific and Industrial Research and the Atomic Energy Board, Pretoria, South Africa.

There has been little work elucidating possible regulatory mcchanisms controlling dark CO, fixation, which we have found can be regulated experimentally. We utilized this finding to begin to determine the nature of the control of heterotrophic CO, fixation in Euglena. We reasoned that the transient elevations of dark CO, fixation observed during growth cycles might be controlled by growth-induced changes in the extracellular medium. MATERIALS AND METHODS Euglena gracilis strain SB3 (permanently bleached by streptomycin) was kindly supplied by Dr. W. R. Evans, Charles S. Kettering Laboratory. Strains Z and SB3 of E. gracilis were grown as described previously (7, 8) in a mineral medium modified from the low p H autotrophic medium of Hutner et al. ( 3 ) . T h e major components of this medium are, in mg/ml: KH2P04, 0.15; MgS0,*7H20, 0.3; MgCO,, 0.3; CaCO,, 0.02; EDTA, 0.2; NH4Cl, 0.3. The carbonates were dissolved in concentrated HCl before addition. The trace metals were added as described previously ( 3 ) , and the p H adjusted to 3.3. This medium is referred to in the text as “mineral medium.”

Cultivation of Leishmania donovani and Leishmania braziliensis in defined media: nutritional requirements.

J. PROTOZOOL. 2 4 ( 3 ) , 437-441 (1977) Cultivation of Leishmania donovuni and Leishmania braziliensis in Defined Media: Nutritional Requirements RO...
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