J. PROTOZOOL. 24(2) , 325-329 (1977)

New Culture Medium for Maintenance of Tsetse Tissues and Growth of Trypanosomatids*

ISABEL CUNNINGHAM? Centre for Trofiical Veterinary Medicine, University of Edinburgh, Easter Bush,

Koslin, Midlothian, Scotland, U.K.,S and Department of Zoology, University of Massachusetts, Amherst, M A 01003, U.S.A.

SYNOPSIS. A new culture medium (SM), based on the amino-acid composition of tsetse hemolymph and containing fetal bovine serum, was designed for the maintenance of tsetse organs and the cultivation of various trypanosomatids. For optimum growth 20% (v/v) serum was required. The medium supported prolonged peristalsis of the alimentary tract and salivary glands of pre-emerged Glossina morsitans morsitans. In established cultures, derived from bloodstream forms of pleomorphic Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense strains, inocula of - 10" procyclics/ml yielded 4-5 x 10' organisms/ml after 4 or 5 days of incubation at 28 C. Bloodstream forms of a cloned monomorphic T . b . brucei strain were also able to transform into procyclics, which, however, multiplied at a lower rate, with maximum yields of - 2 x lo7 after 5 days. Cultures of Trypanosoma congolense and of a nearly monomorphic Trypanosoma brucei gambiense strains could be established in SM medium only in the presence of tsetse alimentary tract. The procyclic trypomastigotes of these species, adapted to SM medium and able to grow in it without Glossina organs, gave maximum populations of - 4.5 x lo' cells/ml.

Promastigotes of Leishmania donouani, cultivated routinely in a diphasic Tobie's medium, multiplied actively upon being transferred into SM medium, producing yields of c 4 X lo7 cells/ml.

Index Key Words : New culture medium; Glossina organ cultures ; Trypanosoma brucei brucei pleomorphic and monomorphic strains; Trypanosoma brucei rhodesiense ; Trypanosoma brucei gambiense; Trypanosoma congolense; bloodstream-to-procyclic form transformation; Leishmania donouani promastigotes.

F the 3 media that have been available to date for the 0 cultivation of the tsetse organ-trypanosome system (4, 21, 2 2 ) , 2, that of Trager (21) and its modification recommended by Cunningham ( 4 ) , contained pupal extract. Even the simpler modified Eagle's medium supplemented with 20% (v/v) sheep serum, more recently devised by Trager ( 2 2 ) , was enriched with pupal extract before flagellates were transferred into it from a primary culture. In all instances, the need for the extract limited the usefulness of the media for growing large numbers of the protozoa required for biochemical and some immunologic studies. To overcome this difficulty, an attempt was made to devise a medium which in the absence of pupal extracts would: ( a ) allow for the maintenance of healthy tsetse organs; ( b ) support large populations of trypanosomatids; and ( c ) provide a suitable en- vironment for serial transfers of these parasites.

The observations of salivarian trypanosomes in the hemolymph of naturally (8) and experimentally (13, 15) infected Glossina suggested to Cunningham & Slater (5) that an analysis of the amino-acid composition of tsetse hemolymph might provide a guide for the design of a new culture medium for tsetse tissues and tsetse-borne parasites. This report deals with results of the attempts to cultivate tsetse organs and various trypanosomatids in a medium in which the amino-acid composition is based on that found in the hemolymph of adult Glossina morsitans inorsitans ( Westwood) (5 ) .

MATERIALS AND METHODS

Culture M e d i u m

The composition of SM (semi-defined maintenance) medium and its method of preparations are given in Table 1 and in the Results.

* This investigation was supported by Research Grants from the Overseas Development Administration of the British Foreign and Commonwealth Office, and A1 00742-20,2 1, from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service.

+From March 1976 to January 1977, the author was a Faculty Research Fellow at the Department of Zoology, University of Massachusetts, Amherst, MA 01003, U. S. A. * Permanent address to which reprint requests should be sent.

A11 compounds, except inorganic salts which were purchased from British Drug Houses Ltd., Poole, Dorset, U.K., or Fisher Scientific Company, Pittsburgh, PA, U.S.A., were supplied by Sigma, and BME (Basal Medium Eagle's) vitamin mixture and the fetal bovine serum by Flow Laboratories.

Trypanosomatid Strains

Trypanosoma brucei brucei Plimmer & Bradford [TREU ( Trypanosomiasis Research Edinburgh University) 11931 was ob- tained in 1974 by injecting infected salivary glands from G. m. morsitans into a mouse. Eightcen days postinoculation the in- fected blood was cryopreserved. One of the stabilates underwcnt three 3-day pasages in mice, and stabilates of this pleomorphic population were usrd to initiate cultures.

Trypanosoma b. hrucei [ETat (Edinburgh Trypanoioon anti- genic type) 31 is a cloned, apparently monomorphic population, very virulent for mice. Its history was described by Lumsdcn and Herbert (11) . Trypanosorncs from a 2-day infection in a mouse were used for cultivation.

Trypanosoma b. gambiense Dutton ( T R E U 1309) w'is isolated in 1967 from a patient at Kwaya Tera sleeping sickness focus in Nigeria by inoculation of lymph gland material into a rat. Srtbse- quently, the trypanosomes were serially subpassaged in 23 adult rats, 23 nursling rats, and 2 nursling mice bcforc beinq adapted and stabilated in mouse blood in 1970. Stabdates of this pre- dominantly monomorphic strain were cultured.

Trypanosoma b. 1 hodesiense Stephens & Fanthani [EATRO (East African Trypanosomiasis Research Organi~ation) 18951 was obtained from a male patient at Lugala, Uganda in 1971. A stabilate prepared from the 7th mouse passage of trypaiiosomes derived from EATRO 1895 stabilate was uscd for cultivation.

Trypanosoma congolense Broden ( T R E U 1183) was isolated from an infected G. pallidipes in Lugala, Uganda in 1970. I t has subsequently been transferred 10 times in mice and then inocu- lated into a rat. Infected rat blood was used to initiate the cul- tures. T R E U 1273 strain of T. congolense was isolated from a naturally infected cow in Uganda in 1960. The trypariosomcs were passaged 3 times in mice, into another cow, then after 3 passages in mire they were inoculated into a rat. Blood from the infected rat was used to initiate the cultures.

325

326 T S F , T S E - T K ~ P A N O S O h f A T I D h'fEDIYh1

Leishmania donocani Laveran Nr Mesnil strain 3s \\-as isolated from a patient in hfalakal area of Sudan in 1966 (see Ref. 17 ) . I t has been maintained in hamsters since that time.

TABLE 1. Coniposition of S M medium.* --

Compound Mg/lOO nil+ Compound Mg/ 100 mlt

Cultication Procedures

I n all instances, the tsetse organs and the trypanosomatids were cultivated a t 28 C either in Lrighton tubes (henceforth rcferred to as tubes) or in 25 cm2 Falcon flasks (henceforth referred to as flasks). On occasion 75 cni2 Falcon flasks were used for growing very large numbers of parasites from established cultures in the absence of tsetse organs. T h e numbers of trypanosomatids \\-ere estimated ivith the aid of a Neubauer heniocytonirter; only motile organisms ivere counted.

Tsetse Organ Cultures

Ne~vly deposited (;lossina tn. ttiorsitans pupa(' supplied by the Tsetsc Research Laboratory, Bristol, were placed in pots of sterile sand and incubated at 28 C, \vith a relative humidity ranging from 65 to 80%.

Puparia containing pharate flies immediately before emergence were surface-sterilized to 10% ( v / v ! Roccal (Bayer Product Co.) for 10-15 min. They w x t ' washed several times in insect balanced salt solution (IRSSj (10; containing 100 1 U / m l penicillin, 100 pgiml streptomycin, and 10 p..g/iiil Furigizone for tissue culture (Squjbb 1 . The \cashed puparia were transferred to a petri dish containing IBSS. Complcte alimentary tract and head with attached salivary glands \\-err removed from the flies and placed in culture medium spread a s a thin film on 9 X 35 mm covw glasses which \\'?re then inserted into tubes. A volume of - 0.5 nil culture medium \\as added to the tubes, each of \chic11 contained the dig.esti\c tract from 3 flies or the salivary glands from 5 flies. The cultures \vere incubated a t 28 C, and the mediuni Lvas changed every 3 or 4 days.

Tr ypan osoma!id Cu It u rc .f

For the cultivation o f T r s j ~ a n t i s t i m a h. b r u c r i ( T R E U 1193) arid 7'. h. rhodrsicnsc ( E A T R O l895), 0.1 nil of infected blood from a stabilatc \\-as diluted \vith culturt, medium to give a concentration of 2-3 x 106 cells/ml, then the try-panosonic sus- pension Ivas placed i n a flask. A s the bloodstream trypanosonirs transformed into procyclic forms and multiplied, culture medium as added u p to a volume of L, nil. When the population reached - 2 x IO7 cc.lls/nil, the supernatant fluid free of blood cells was transferred into flasks containing 3 nil of SM to give an initial concentration of - 3 x 10'; cellsjml. Thereafter the organisms were passagid twice \\-cekly, u i t h initial inocula of - 106 cells/rnl.

l 'rypdnosovia b. hrucci ETat :i j cultures were initiated by mixing infected blood with Sh4 to give a concentration of 4-5 X 106 cellsjnil; 1.0 nil of the trypanosonir suspension \\as placrd in a flask. On thc 4th day, ivhen the trypanosomes were beginning to transforrii into slendrr procyclics, an equal volunie of medium \\-as addcd. ;'\ftt.r 4 morc days, the flagellates \ \ere transferred into flasks at an initial concentration of - 1.5 X I U t i rclls/ml and were thrrcaftrr subcultured every 5 or 6 days a t conccntrations of 1-2 x 10": nil.

C1ulture.c of Trybanosotna h. gambiense ( T R E U 1309 ) and T . rongolen.re ( T R E Y I183 and 1273) \\-ere started by adding bloodstreani trypnnonomrs to SM to a coiiccritration of 2-3 X 106 cells~tnl. Aliquots of 0.25 nil of the suspension were placed in tubes containing freshly dissected alimentary tracts from S pre- emerged G. m. morsitnns. On the 4th or 5th day when the organisms transformed fully- and multiplied, 0.3 nil of culture

Inorganic salts NaHaP04 MgCli . 6HzO MgSor 7HzO KCI (anh.) CaCL 2 H L 0

Glucose Fructose Sucrose

Organic acids L-Malic a-Ketoglutaric F u m a r i c Succinic

.\mino acids p-Alanine

u~-.4lanine ~-Arginine ~-.4sparagine ~-.4spartic acid L-Cysteine HCI

Sugars

53 L-Cystine 3 304 L-Glutamic acid 25 370 L-Glutamine 164 298 Glycine 12

15 1.-Histidine 16 i>L-Isoleucine 9

70 L-Leucine 9 40 L-Lysine 15 40 uL-Methionine 20

L-Phenylalanine 20 67 L-Proline ti90 37 nL-Serine 20

5.5 L-Taurine 21 6 ix-Threonine 10

L-Tryptophan 10 200 L-Tyrosine 20 109 ix-Valine 21 44 Vitamin mixture

11 Indicator 24 BME ( loo><) 0.2 ml

8 Phenol red (0.5%) 0.4 ml

* For method of preparation, see Results. Fetal bovine serum a t the final concentration of 20% (v /v) is added before use. Osmo- lality of the freshly prepared complete medium (with fetal bovine serum) = 304 miliosnioles/kg. pH 7.4, adjusted with 2 N NaOH.

t Except as indicated.

niediuiii MYIY added to each tulic. Aftcr a further 4-day period, the rich population of trypanosornes was transferred from a tube into a flask, care being taken to leave behind thc blood cells most of \\-hich adhrrcd to the bottom of the tubes. T h e cells in the flask were counted, and a sufficient volume of fresh me- dium \\-as added to give a concentration of - 4 X lo6 cells/ml. If t h r volume of this trypanosome suspension was less than 3 nil, a small amount of fresh medium was added to the growing population until the volume reached 3 nil. Inocula of 3-4 X lo6 cells/ml were used for the immediately following 2 subcultures. Thrreafter, the "adapted" procyclics were maintained in flasks \\-ith initial concentrations of - 106 cells/ml.

Cultures of Lrishwania donovani strain 3s were prepared using established promastigotes grown in Tobic's medium ( 2 0 ) . T h e flagcllates \\ere gro~vn in flasks containing 3 nil SM.

RESULTS

Cuiturc Medium

T h e salt, sugar, and organic acid composition of SM medium, shoirn in Tablr 1, is a modification of Grace's (9) medium. Its amino acid constitution is based upon that of tsetse hernolymph as reported by Cunningham Nr Slatrr ( 5 ) .

T h e mrdiuin \\-as prepared by dissolving the compounds in 2X distilled water as folloivs: ( a ) inorganic salts, except for CaCI,, in 10 inl; CaCI, in 5 nil; ( b ) sugars in 10 ml; ( c ) organic acids in 5 nil; ( d ) amino acids in 65 inl. T h e several solutions were mixrd and thc pH kcas adjusted to 7.4 with 2 N NaOH. The mixture !\-as sterilized by filtration, using a Millipore Sterifil apparatus with a 0.22 pn i filter. It was then divided into cori- venicnt volumes and stored at -20 (2 until needed. Before use, the nicdium was thawed, and either noninactivated or hrat-inac- tivated fetal bovine was added to a final coticentratiori of 20%

The S M medium not only supported long-trrm maintenance of tsrtse organs, but was also suitable for the establishment and g r o w h of certain trypanosomatids. As is shown in the results

( v i v ) .

TSETSE-TRYPANOSOMATID MEDIUM 327

(see below), the establishment of cultures of some trypanosome strains required the presence of tsetse organs, while cultures of other strains could be initiated without those tissues.

Tsetse Organ Cultures

The crop duct, midgut, Malpighian tubules, and hindgut in explants of complete alimentary tract contracted vigorously for 2 weeks. Less active peristalsis of the midgut persisted for a further 10 days; in a few of the cultures the contractions were noted for up to 40 days. Salivary glands from flies of the same age contracted rhythmically for 9 or 10 days in SM.

Trypanosomatid Cultures

Trypanosoma b. brucei ( T R E U 1193) and T. b. rhodesiense (EATRO 1895) behaved in a similar manner in SM medium. With inocula of 2-3 x 106 bloodstream forms/ml, a lag phase lasting - 24 hr waq followed by a period of transformation into procyclic trypomaqtigotes and growth. On addition of fresh medium to a total volume of 2-3/ml, the concentration of the cells increased to - 2 x lo7 after 4 or 5 days. The first sub- cultures with inocula of - 3 X 106/ml gave rise to populations of - 3 XlO7 in 3 or 4 days. Subsequent passages, with smaller inocula, yielded larger populations as shown in Table 2. Similar counts were recorded for T . b. rhodesiense.

There was no difficulty in growing T. b. brucei ( T R E U 1193) in large volumes of SM, e.g. yields of - 4 X lO7/rnl were ob- tained in 65-ml volumes in 75 cm2 Falcon flasks. The yields were unaffected by continuous cultivation of up to 3 months, at which time the experiment was discontinued and the trypano- somes were cryopreserved.

Investigation of the effect of the fetal bovine serum concen- tration in SM on growth of T. b. brucei ( T R E U 1193) indi- cated that, in the presence of 20% (v/v) serum, an initial inoc- ulum of - 1O6/ /ml resulted in a population of - 4.6 X 107/ml after 3 days. Lower yields were obtained with comparable in- ocula in SM containing 10% (v/v) serum, the counts being only - 3 X 107/ml after 4 days. Some cells in the latter cultures ap- peared to be less actively motile than those grown in the pres- ence of 20% serum.

Bloodstream trypanosomes of monomorphie T. b. brucei (ETat 3 ) had multiplied and transformed into procyclic forms by the 6th day in culture. Growth of the newly transformed procyclics, however, was slow during their first transfer; an initial concentlation of - 1.5 X 106/ml reached - 7 X 107/ml after 7 days. Subsequent passages with the same inoculum multi- plied at a faster rate and yielded populations of - 2 X 107 in 5 days.

Culture medium alone did not support the transformation of T. congolense ( T R E U 1183 and 1273) bloodstream forms into procyclic trypomastigotes. This transformation required the presence of tsetse alimentary tract. After a short lag phase, the parasites transformed and multiplied, a population of - 3 x lO7/rnl being reached by 5 days in the primary cultures. The addition of 2 volumes of SM to the culture tubes led to a further increase in the trypanosome numbers to - 4 X 107/ml on the 7th or 8th days. When the procyclics were transferred to flasks and cultivated in the absence of tsetse tissues, it was necessary to use initial inocula of 3-4 X 106/ml for the first 2 or 3 passages. These inocula produced populations of - 4 x lO7/ml after 4 days. After this stage, the procyclics were re- garded as fully adapted to SM and subsequent cultures were initiated with inocula of - 106/ml. Maximum yields of - 4.5 x 107/ml were obtained in 4 days, and the cultures were main- tained for 3 months with no change in the yields or growth rates.

TABLE 2. Growth of procyclic T. brucei strain TREU 1193 during routine maintenance in 3 ml volumes of SM medium in 25 cm’

Falcon flasks. - -

Nos. ( x 10‘ per ml)

Day Mean -C SE Mean 5 SE

0 0.5* - 1.2* - 1 1.9 & 0.45 3.7 I+ 0.24 2 4.3 -t 0.26 11.1 f 1.81 3 26.8 C 0.81 43.0 2 1.33 4 52.1 C 0.77 52.7 f 0.95

52.8 2 0.53 5 6 37.1 & 1.8 40.3 f. 1.07

53.2 C 0.74

* Initial inoculum.

A marked decrease in p H of the medium was noted during the exponential growth phase of established T. congolense cul- tures, the initial p H of 7.4 dropping to 6.4 after 3 days. The addition of 10 or 20 mm HEPES to SM medium did not aid in stabilizing the p H during the logarithmic growth phase of the procyclics. I t was observed also that T. congolense pro- cyclics were able to tolerate higher p H values, e.g. up to 8.2, without any apparent adverse effects upon the cell yields. De- creases in pH, similar to those accompanying logarithmic growth of T. congolense were evident also in media with actively multi- plying procyclics of T . b. brucei strain TREU 1193 and of T. b. rhodesiense.

The initiation of primary cultures of T . b. gambiense ( T R E U 1309) with bloodstream forms required the presence of tsetse alimentary tract. In such cultures the parasites followed a pat- tern of growth and transformation similar to that already de- scribed for T. congolense. During the first 2 or 3 subcultures in SM in the absence of tsetse tissues, inocula of - 3 X lo6 cdls/ ml gave rise to populations of - 2 X 107/ml after 4 days. There- after the established procyclics multiplied at a faster rate, yield- ing populations of slightly more than 4 X l07/ml from inocula of - 106/ml during the same period.

The following observations pertain to the primary cultures of T . congolense (TREU 1183) and T . b. fiambiense (TREU 1309). When the procyclics of these species were removed from the tubes and transferred into flasks, the tsetse alimentaty tracts in the tubes were washed with SM to remove red cells and then covered with - 0.5 nil of culture medium. The procyclics which remained in association with the tsetse organs began to multiply, and by 10-12 days after initiation of the cultures, var- ious regions of the digestive tract became invaded by the flagel- lates. On occasion, proventriculi, midguts, hindguts, and Mal- phigian tubules were packed with the trypanosomes, a situation resembling that reported previously by Trager (21) . Also, as indicated by that author (22), these invasions appeared to have little effect on peristalsis of the organs. The forms of trypano- somes found on a careful examination of the Giemsa’s-stained preparations of triturated, invaded tsetse organs rescmbled those found in the midgut and the proventriculus of Glossina.

Leishmania donouani strain 35 promastigotes established in Tobie’s medium (20) grew well in SM at 28 C. Inocula of - 1.5 X lo6 cells/ml gave rise to cultures with maximum popula- tions of - 4.5 x l07/ml in 4 days.

DISCUSSION

In organ cultures of arthropod digestive tract the survival of the tissues is generally estimated by the maintenance of con-

328 TSETSE-TR~.ANOSOhIATID bfEDIWh1

tractions (for pertinent references, see Ref. 6 ) . Peristalsis of the alimentary tract of pre-emerged G . 7 n . morsitans continued for several weeks and the salivary glands contracted for many days in SM culture medium. Rhythmic contractions of the gut persisted even when it was invaded by the parasites and also in the presence of rapidly growing populations of trypanosomes.

It has been customary to grow salivarian trypanosomes in media which do not contain multiplying vertebrate or inverte- hrate cells [see the review by Bishop ( 2 ) ] . Most of those media contain relatively large quantities of blood, as in original mono- phasic solid NNN (14:, diphasic Tobie’s (20) , and the mono- phasic liquid medium of Pittam ; 16) . More recently, defined and semidefined liquid media in \vhich the blood has been re- placed by hemiri and other growth factors have been described by Cross & Manning ( 3 ) and Steiger (18).

A chemically complex medium incorporating tsetse pupal extracts, as originally devised by Trager (21 ) or as slightly mod- ified by Cunningham (4), ivas used successfully for the culti- vation of various salivarian species. Transformation into pro- cyclic or procyclic-like forms and good growth in these media, howwer, occurred only in the presence of tsetse organs. The cultures were maintained in small volumes of media in very small flasks C J ~ in hanging drops, Lvhich iniposcd limitations on the total yields of the parasites. After having been established in the presence of Glossina tissues, Trypanosoma uicax could he groxvn on NNN slants, hut only quite small populations of the flagellates were obtained by these means ( 2 1 ) . In both the primary cultures and in subcultures, the stages of T. b. brucei and T. congolense morphologically similar to those found in the vector \vere observed, although not necessarily in their normal proportions; in T. cimx culturea, midgut- or proventricular-like trypomastigotes. not knon.n from G[o.r.tina, \verc also notcd. Subsequently, Trager (22) used a modification of Eagle’s ME medium for the cultivation of T. vivax. The medium was modi- fied by beinx strongly buffered and supplemented with 20% ( v / v ; sheep serum. As in the previous methods (4, Z l ) , the total volumca of the cultures Ivere quite small. The primary cultures consisted first of c,longate procyclic-like forms, then of typical epimastigotes; transitional forms rvith lateral and pos- tcrior kinetoplasts \vere found in older cultures (22) . Epimasti- gotes predominatrd upon transfer into new medium.

The medium described in the present report provided for wry large yields of T. b. brucei, T . b. rhodesiense, and T . con- golense. The presence of Glossina organs was required only for the rstablishment of the primary cultures of T. congolense and T. ganzhirnse. Even these species, however, could be grown on subsequent passqes in SM without tsetse tissues and large parasite yields v.‘rre obtained under these conditions.

Thp yields of procyclics of T. h. brucei ( T R E U 1193), T. gnirthieiise, T . h. rhodesirnse, and strains of T . congolcnse grown in SM medium compared favorably ivith those reported for thcw spwies cultivated in other media. According to Tobie et al. ( 2 0 ) , their diphasic nirdiuni, which has been used widely for the last quarter of a century for the culti\ration of Salivaria, inoculated \\ith 5 x lO5,’ml hloodstream T. b. gainbiensc and T . h. rhodcsienw produced 1.4 x lO7/ml procyclic forms in 7 days. Liquid mrdia havc many advantages over diphasic ones; above all? the study and harvesting of the cells is facilitated. I n thc hlood-lysate liquid medium of Pittam (16) initial con- centrations nf 3 X 10”ml hloodstream T. b . rhodesiense gave rise to 3 x 10; flagcllates;ml after 4 days. Slightly superior yields of u p t o 4 X 107 cellsjml of T. brucci \\-ere obtained in the semisynthetic medium of Steiger (18) , hut the chemically

defined medium of Cross & Manning ( 3 ) produced much lower populations ( - 7.4 X 106/ml) of T. b. brucei procyclics.

The inability of T. b. gambiense and T. congolense blood- stream forms to develop into procyclics in SM medium without tsetse tissues is not fully understood. The use of a frozen sta- b i k e of a predominantly monomorphic strain of T. b. gam- biense may have contributed to its inability to transform and multiply in the medium alone. The growth factor or factors produced by the living gut in the primary cultures may be related to these which supported the multiplication of T. b. brucei in association with Glossina alimentary tract in modified Trager’s medium (4 j .

Of interest are the very recent findings on the development of procyclics in cultures of the cloned monomorphic strain of T . b. brucei ETat 3. The transformation of this strain from bloodstream to procyclic forms was prolonged over a period of 5 or 6 days, as compared with the 2-3 day period of the other species grown in S M medium. Admittedly, a t least during the early transfers performed to date, the maximum yields of this strain in S M medium were smaller than those recorded for the pleomorphic trypanosomes. These results appear to be at vari- ance with the numerous published observations according to which true mononiorphic strains were neither cultivable nor capable of producing mature infections in tsetse flies.

Many of the blood-enriched media used for the cultivation of salixrarian trypanosomes have also been found suitable for the growth of Leishmania spp. The monophasic liquid medium devised by Dwyer ( 7 ) supported luxuriant growth of L. dono- m n i , producing ,- 7.2 x 106 promastigotes/ml after 7 days. Another liquid medium, formulated by Mansour et al. (12), in which the amount of blood was reduced to 10% (v/v) , p:o- vided yields slightly superior to those obtained from Tobie’s diphasic medium which contains 30% defibrinated blood. Re- cently, Berens et al. ( 1 ) developed a semidefined liquid medium, based on Eagle’s Minimal Essential Medium, which was able to support good growth of I,eishmania spp. and of Trypano- sorna cruzi. The medium described in the present report pro- duced comparable yields of Leishmania promastigotes, but no attempt was made to establish the cultures from amastigote forms. O n the other hand, a better growth of L. donovani promastigotes was obtained in the defined medium of Steiger & Steiger ( 19), but the high counts apply to the stationary phase.

ACKNOWLEDGEMENTS

It is a pleasure to thank Mrs. Janis Pringle for her excellent technical assistance and Professor B. M. Honigberg for valuable advice during the preparation of this manuscript.

REFERENCES

1. Berens RL, Brun R, Krassner SM. 1976. A simple mono- phasic medium for axenic culture of hemoflagellates. ]. Parasitol.

2. Bishop A. 1967. Problems in the cultivation of some parasitic protozoa, in Dawes B., ed., Aduances in Parasitology, Academic Press, New York 5, 93-138.

3. Cross GAM, Manning JC. 1973. Cultivation of Trypano- J O W I U brucei sspp. in semi-defined and defined media. Parasitology 67. 315-33.

4. Cunningham I. 1973. Quantitative studies on trypano- somes in tsetse tissue culture, E x p . Parmitol. 33, 34-45.

5. Cunningham I, Slater JS. 1974. Amino acid analyses of haemolymph of Glossina morsitans morsitans (Westwood). Acta Tropica 31. 83-8.

6. Demal J, Leloup AM. 1972. Organ culture of insects, in Vago C., ed., Invertebrate Tissue Culture, Academic Press, New York 2, 3-39.

62, 360-5.

TSETSE-TRYPANOSOMATID MEDIUM 329

7. Dwyer DM. 1972. A medium for cultivating Leishmania donovani in large numbers. J. Parasitol. 58, 847-8.

8. Foster R. 1963. Infection of Glossina spp. Wiedemann 1830 (Diptera) and domestic stock with Trypanosoma spp. Gruby 1843 (Protozoa) in Liberia. Ann. Trop. Med. Parasitol. 57, 383-96.

9. Grace TDC. 1962. Establishment of four strains of cells from insect tissues grown in vitro. Nature, London 195, 788-9.

10. Jones BM, Cunningham I. 1961. Growth by cell division in insect tissue culture. Ex$. Cell Res. 23, 386-401.

11. Lumsden WHR, Herbert WJ. 1975. Pedigree of the Edinburgh Trypanosoma ( Trypanotoon) Antigenic Types (ETat) . Trans. Roy. SOC. Trop. Med. Hyg. 69, 205-8.

12. Mansour NS, Hady J, McConnell E. 1973. A modified medium for Leishmania. J. Parasitol. 59, 1088-90.

13. Mshelbwala AS. 1972. Trypanosoma brucei infection in the haemocoel of tsetse flies. Trans. Roy. SOC. Trop. Med. Hyg.

14. Novy FG, MacNeal WJ. 1904. On the cultivation of Trypanosoma brucei. J. Infect. Dis. 1, 1-30.

15. Otieno LH. 1973. Trypanosoma (Trypanozoon) brucei in the haemolymph of experimentally infected young Glossina morsitans. Trans. R . Soc. Trop. Med. Hyg. 67, 886-7.

66, 637-43.

16. Pittam MD. 1970. Medium for in uitro culture of Trypanosoma rhodesiense and T. brucei, Appendix (pp. 127-8), in Dixon H, Williamson J. The lipid composition of blood and culture forms of Trypanosoma lewisi and Trypanosoma rhodesiense compared with that of their environment. Comp. Biochem. Physiol.

17. Stauber LA. 1966. Characterization of strains of Leish- mania donovani. Exp. Parasitol. 18, 1-11.

18. Steiger R. 1976. A new culture medium for Trypano- soma brucei. J . Prototool. 23, 33A-4A.

19. ___ , Steiger E. 1976. A defined medium for culti- vating Leishmania donovani and L. braziliensis. J. Parasitol. 62, 1010-1.

20. Tobie EJ, von Brand T, Mehlman B. 1950. Cultural and physiological observations on Trypanosoma rhodesiense and Trypanosoma gambiense. J. Parasitol. 36, 48-54.

21. Trager W. 1959. Tsetse fly tissue culture and the De- velopment of trypanosomes to the infective stage. Ann. Trop. Med. Parasitol. 53, 473-91.

22. ~ 1975. On the cultivation of Trypanosoma vivax: a tale of two visits in Nigeria. J . Parasitol. 61, 3-1 I .

33, 111-28.

J. PROTOZOOL. 24(2) , 329-331 (1977).

Extra Nutritional Requirements of Artificially Aposymbiotic Crithidia deanei*

MARIA HERMELINDA MUNDIM and ISAAC ROITMAN Depar’tamento de Biologia Celular, Znstituto de Cizncias Bioldgicas,

Universidade de Brasilia, Brasilia, DF, B r a d

SYNOPSIS. Chloramphenicol cured Crithidia deanei of its endosymbiote. The derived aposymbiotic strain had additional growth requirements: purin (as adenine), heme, arginine, histidine, isoleucine, leucine, phenylalanine, threonine, trypto- phan, valine, pyridoxine, riboflavin, and pantothenate and liver infusion (replaceable by high nicotinamide) .

Index Key Words: Aposymbiotic Crithidia deanei; growth requirements.

RYPANOSOMATIDS harboring endosymbiotes, Crithidia T oncopelti and Crithidia deanei, can be grown in simple defined media (7, 8) . Attempts with several antibiotics to “cure” C. deanei failed (7) . Recently, symbiote-free strains of Blastocrithidia culicis and C. oncopelti were obtained with high doses of chloramphenicol ( 3 ) ; by a similar method we secured an aposymbiotic strain of C. deanei. In the present paper we describe the nutrition of this aposymbiotic strain.

MATERIAL AND METHODS

Organisms.-Crithidia deanei ( ATCC 30255) from Zelus leucogrammus ( 1 ) was maintained in a simple defined medium ( 7 ) . To obtain the cured strain, C. deanei was inoculated into a blood-broth consisting of: NaCl, 1.48 g; peptone (Difco), 5.4 g; H20 450 ml; defibrinated rabbit blood 5% (v/v). The medium was distributed in 5-m1/18 x 180 mm screwcap tubes; 0.4 pg/ml chloramphenicol were added aseptically. After 3 transfers at 28 C at 15-day intervals, the simple defined medium no longer supported growth. No endosymbiote could be dem- onstrated by electron microscopy. The aposymbiotic culture was initially maintained in blood-agar with a NaCl 0.85% over- lay, later in a defined medium for Herpetomonas samuelpessoai (9) with liver infusion (Difco) 0.4% (w/v). The strain is now

* This investigation was supported by the National Research Council of Brazil. We thank Dr. S. H. Hutner for help with the manuscript.

maintained in the same defined medium with 3.0 mg% (w/v) nicotinamide replacing the liver infusion.

Growth Experiments.-Experimental media were distributed in 5-ml volumes to 18 X 180 mm metal-cap tubes, and auto- claved at 118-121 C for 20 min. Each tube received 1 drop of a 48-72-hr culture grown at 28 C. In all experiments incubation was at 28 C. Growth was measured in a KIett-Summerson photo- colorimeter with a green filter, and scored in Klett units. Nu- tritional requirements were examined by the omission technic. For identification of vitamin requirements successive transfers were carried out.

RESULTS

In the defined medium for H. samuelpessoai (9) the aposym- biotic C. deanei had a residual growth but no longer grew in the defined medium described for the symbiote-harboring parent strain ( 7 ) . Increase of the liver infusion content was accom- panied by a proportional increase in growth. Optimal growth was achieved with 0.4% (w/v) liver infusion; growth was in- hibited with higher concentration of the infusion (Table 1 ) .

In an attempt to replace liver infusion each constituent of the defined medium (9) was tested. Only nicotinamide elicited stimulation, optimally a t concentrations of 3-5 mg% (w/v) (Table 1 ) .

There were many nutritional differences between the aposym- biotic C. deanei and its parent (Table 2 ) . Like the latter ( 7 ) , the aposymbiotic strain grew a t 15-34 C, with optimum at 28-