Cytoplasmic inclusions and organelles of in vitro cultured Trypanosoma theileri and Trypanosoma melophagium and some speculations on their function

EXPERIMENTAL

17, 24-40

PARASITOLOGY

Cytoplasmic Cultured

(1965)

Inclusions

and

Trypanosoma

melophagium

theileri and

for

in

Vitro

Trypanosoma

Speculations

on

Herbert

Agricultural and Forest of North Wales, Bangor,

(Submitted

of

Function

I. V. of

and

Some

Their

Department

Organelles

publication,

Zoology, University Great Britain 26 May

College

1964)

HERBERT, I. V. 1965. Cytoplasmic inclusions and organelles of in vitro cultured trypanosoma theileri and Trypanosoma melophagium and some speculations on their function. Experimental Parasitology 17, 24-40. The cytochemistry and fine structure of inclusions and organelles of Trypanosoma theileri and Trypanosoma melophagium have been studied. One or both of these techniques has permitted the demonstration of mitochondria, lysosomes, ribosomes, endoplasmic reticulum and Golgi apparatus. The phagosomes (digestive lysosomes) demonstrated by cytochemical techniques are discussed in relation to the probable pinocytotic feeding mechanism of the trypanosomes studied, and analogies between the lysosomes of vertebrate cells and trypanosomes are suggested. An attempt is made to characterize the above organelles by histochemical means, not only to ascertain their chemical affinities but also to demonstrate their affinity to some descriptions of ‘volutin’ bodies seen in other trypanosomidae. From the available evidence it seems likely that previous descriptions of volutin in trypanosomidae have described either several differ__ ent inclusions or organelles, or inclusions or organelles at differing stages of development and activity.

Some aspects of the cytochemistry of in vitro cultured Trypanosoma theileri Laveran, 1902 as seen under the ordinary light microscope have ‘been dealt with by Herbert (1965), and it was shown that a number of cytoplasmic bodies reacted positively with dyes designed to identify neutral lipid, phospholipid, ribonucleic acid, and polyphosphate; but it was impossible to determine whether these dyes were bound on to inclusions or organellesor whether different types of inclusions or organelles existed. Phase-contrast and dark-field illumination microscopestudies had not suggestedthat more than one type of cytoplasmic body was present, though Giemsa-stained preparations and supravital staining suggested there was. The rapidity 21

with which neutral red and Janus green B dyes were absorbed into the living trypanosomesbody to locate on some or all of the above cytoplasmic bodieswasnoted, and it was suggestedthat this might be due to an active phagocytosis or pinocytosis involving the agency of phagocytotic or pinocytotic lysosomesor lysosome-like bodies. Some further speculation was made concerning the possible identification of other organelles, though the tests performed were not specifically designed to identify them. An attempt was made to characterize and compare the so-called ‘volutin’ bodies of T. theileri with those described by various authors working on other micro-organisms. The accumulation of volutin is a constant

CYTOPLASMIC

INCLUSIONS

feature of in vitro cultivated T. theileri and indeed in virtually all trypanosomidae whether grown in vitro or in viva, volutin bodies being most readily demonstrable under the ordinary light microsocope in organisms which are suffering a crisis in numbers. It was decided to investigate the nature of the inclusions and organelles further, utilizing histochemical studies with the ordinary light microscope. This was done by removing organisms from cultures at daily intervals to determine what changesin reactivity occurred in the cytoplasmic bodies during the growth cycle, or which bodies reacted at certain periods of the growth cycle but not at others. On the basis of these findings and observations on the in vitro culture of T. theileri at 28°C (Herbert, 1963), it was decided to attempt to demonstrate specific organelles and enzymes concerned with them, particular attention being paid to those organelles and enzymes that might be concerned in the absorption and utilization of nutrients. The electron microscope was employed to a limited extent in an attempt to detail the structure of the trypanosome cell to clarify some of the histochemical findings. Comparison was made with in vitro cultured Trypansoma melophagium Flu, 1908, since T. theileri and T. melophagium are taxonomically related (Wenyon, 1926). Trypansoma melophagium was isolated by the writer in 1960 from the sheep ked, Melophagus ovinius L. The discussion includes somespeculationson the possiblesignificance of the findings of this paper and a previous paper (Herbert, 1965).

AND

ORGANELLES

25

of multiplication. The organisms were crithidia, though a few true trypanosome forms occurred. The changes in histochemical staining reaction of trypanosomes during their growth cycle in culture were examined as follows: Trypanosomes from 4 culture tubes were mixed on each of 10 successivedays and a sample counted in an improved Neubauer blood-counting slide. The growth curves are detailed in Fig. 1. Harvested trypanosomes were washed twice in physiological saline, concentrated by centrifugation, fixed, and stained. In most instances trypanosomes were mounted onto glass slides or coverslips immediately after fixation and before staining, but it was occasionally necessaryto stain fixed specimensbefore mounting. The glass slides or coverslips onto which the organisms were to be placed were first smeared with a 1: 15 (v/v) concentration of glycerine albumen: water mixture. After this was dried the organismswere placed onto a marked area of the smear. Drying was watched under the microscope until the organisms were sufficiently dry that they adhered to the slide, but before they becamecompletely dried. The preparation was then plunged into distilled water to arrest further drying. Too little drying caused the organismsto be washed off at this point; too much drying led to ultimate nonspecific staining. The histochemical tests employed were those required to distinguish between the chemical affinities of the cytoplasmic bodies. The trypanosomes varied in size according to the fixative used, and this may lead to discrepancieswhere the results of one regime are compared with another. METHODS The tests employed were as follows: All cultures of T. theileri and T. melophaTo&dine blue reaction. The standard gium used in these tests were grown at 28OC method of Pearse (1960) was used after in an enriched N.N.N. medium described by form01 calcium fixation. The toluidine blue the writer (Herbert, 1961). Test tube slope dye was obtained from G. T. Gurr Ltd., cultures were prepared by inoculating the London and was a good sample of metaabove diphasic medium with organismstaken chromatic dye. It was made up in neutral disfrom J-day-old cultures which were in a state tilled water as a 0.5 per cent solution. Stain-

26

HERBERT

ing was always best where the trypanosomes were stained a few hours only after fixation. Control preparations were submitted to digestion in pure crystalline ribonuclease enzyme (0.1~ gm/ml sterile distilled water) for one hour at 37°C. Al&an blue reaction. For acid mucopolysaccharides the method of Steedman (1950) was used after form01 calcium fixation. The dye was obtained from G. T. Gurr Ltd., London. Pyronin-methyl green (PMG.) . For deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) the method of Jordan and Baker (1955) was used after Schaudinn fixation. The specificity of the pyronin for RNA was ascertained by running control tests with pure crystalline ribonucleaseenzyme (Armour Laboratories Ltd., England). The dye was obtained from G. T. Gurr Ltd., London. Sudan black B. Sudan black B in propylene glycol after form01 calcium fixation according to the method of Pearse (1960) was used. The dye was obtained from British Drug HousesLimited, England. Control slides were done by extracting the lipid with pyridine (Baker, 1946). Copper phthalocyanin test. For phospholipid the method of Pearse (1960) was used after weak Lewitsky fluid fixation. The methasol fast blue B dye was a gift from Imperial Chemical Industries Ltd., Manchester, England. Control slides were done by extracting the lipid with pyridine (Baker, 1946). Naphthol AS-BI phosphate method. For acid phosphatase enzyme the method of Pearse (1960) (after Burstone) was used after cold (4’ C) acetone fixation. The redviolet LB salt and the naphthol AS-B1 phosphoric acid were obtained from Sigma Chemicals Inc. U.S.A.

grown as described earlier, washed from cdtures, concentrated by centrifugation, fixed, mounted, and stained as detailed in Table I. All the organisms studied were taken from 3- or 4-day-old cultures since this represented a time of maximum numbers when organisms were easy to harvest. Tests were designed to identify mitochondria and the mitochondrial enzymes, cytochrome oxidase, and succinic dehydrogenase. A previous paper (Herbert, 1965) has described the accumulation of phosphate and polyphosphate which appear as granules within the cytoplasm of T. theileri after fixation and staining, and so tests were run to demonstrate the presence of different phosphatases, viz. alkaline and acid phosphatases, adenosine triphosphatase, alkaline and acid inorgacic pyrophosphatases. Since lipid is also present and because in in vitro culture experiments the absorption of supra-vital dyes and the presence of acid phosphatase enzyme had led the writer to suspectthe presenceof lysosomeor lysosomelike structures in the trypanosome cell, tests were also run for certain enzymes which are (de Duve, 1962) present within vertebrate cell lysosomes,viz., fi glucouronidaseand lipid hydrolysing enzymes. For the same reasons tests were also run for lipofuchsins and phagosomes. The fixatives, if any, and the references to the tests performed are summarized in Table I. All dyes, except where otherwise stated, were obtained from G. T. Gurr Ltd., London.

Trypanosomes to be used in electron microscope studies were harvested 4 or 9 days after cultures were inoculated, washed three times by centrifugation in ice-cold Ringer’s solution, and fixed for 3 minutes at 4OC. in Palade’s ( 1952) buffered osmium tetroxide Besidesthe above tests a number of other fixative. The organisms were massedinto a histochemical examinations was made of the pellet by centrifugation and dehydrated cytoplasmic inclusions and organelles. through graded ethanol/water mixtures to 70 Trypanosomes used in these tests were per cent ethanol. The trypanosomes were

CYTOPLASMIC

INCLUSIONS

transported in this condition to Dr. R. G. Bird at the London School of Tropical Medicine and Hygiene. Dehydration was completed in ethanol, the last change of which contained 1 per cent phosphotungstic acid to improve contrast of sections, embedded in ‘Araldite’ in an oven at 60°C and the resin hardened for 3 days. Sections were cut on a Huxley pattern Cambridge Instrument Co. ultramicrotome, and sections were viewed on carbon-coated grids under a Metropolitan Vickers E.M.6 electron microscope. RESULTS

The changes in the histochemical staining reaction of trypanosomes during their growth cycle in culture are detailed for comparison in Fig. 1, which details the growth curve of T. theileri and T. melophagium. Growth is logarithmic (though there may be an initial lag in growth) until the third or fourth day when a decline sets in. The decline is quicker for T. theileri than for T. melophagium. Sampling ceased on Day 10 since the cultures of T. theileri were very degenerate and accurate histochemical staining was difficult. Cultures which have reached this stage of growth die out within a few days although one may occasionally find a few live cells for some considerable period. The results discussedunder the following histochemical tests apply to both T. theiZeri and T. melophagium except where specifically stated. One can always find atypical forms, some of which contain bodies which do not react with the dyes described. The following remarks refer to the cytoplasmic bodies, some of which appeared as staining granules, others as vacuoles.

AND

27

ORGANELLES

l.o:

.3

5

7

FIG. 1. Growth of T. theileri gium in test tube slope cultures of and nutrient agar. The contents of were mixed and a sample withdrawn ture for counting on each of ten

9

/I

and T. melopha20 per cent blood four culture tubes from this mixsuccessive days.

the nucleus, varied in size, but up to 2 u in diameter. In succeeding days they increased in number and size until by the fourth day they almost filled the cytoplasm so that it was difficult to distinguish between individual bodies. The proportion of bodies staining metachromatically also increased and all the larger ones were stained in this way. No change was detectable until the seventh day when a few non-staining vacuoles appeared, especially in rounded atypical forms. The cytoplasm otherwise remained full of orthochromatic and metachromatic staining bodies. The number of bodieson Day 1 was greater in T. melophagium than in T. theileri, this presumably being accounted for by the lag in growth, but by the second day the numbers had decreased slightly, and both orthochromatic and metachromatic bodies were well defined in an otherwise clear cytoplasm. All aggregations were posterior to the nucleus. The number of bodies did not appear to increase until Day 6 when rounded trypanoTo&dine Blue Reaction somesappearedwhich were full of cytoplasmic On Day 1 the numbers of orthochromatic- bodies, the metachromatic ones being domially and metachromatically stained bodies in nant. Long slender forms had fewer granules. T. theileri were few and irregularly disposed From Day 7 onwards the numbers increased throughout the cytoplasm. If they were ag- until they packed the cytoplasm. No vacuoles and very degregated then they were usually posterior to were seen in T. melophagium,

28

HERBERT

generate trypanosomes were not seen by the tenth day of culture. In both trypanosomes the orthochromatic staining could be removed by digestion in pure crystalline ribonuclease, and this rendered the metachromatic bodies easier to distinguish. The RNA containing orthochromatic bodies were in general smaller than the metachromatic granules though one occasionally seeslarge orthochromatic bodies. Alcian

Blue

Reaction

T. theileri (up to 1 p in diameter), few in number, and rarely aggregated. These increased in number as time progressed until by the sixth day the cytoplasm was massed with small and large (up to 3 p in diameter) inclusions. On Day 1 there were more lipid inclusions in T. melophagium than in T. theileri, but on Days 2 and 3 there were less. These increasedin number but did not pack the cytoplasm until Day 8. Large lipid inclusions (above 2 p in diameter) did not appear till Day 10. No non-staining vacuoles were seen in either organism.

The reactions recorded for the test are regarded as negative for both T. theileri and T. melophagium. An occasional positively Copper Phthalocyanin Test staining body was observed, but it was conThe phospholipid positive inclusions were cluded that this was due to agar-agar connever so numerous as the lipid inclusions seen tained in the culture medium which was not after Sudan black B staining. Where they did washed from the trypanosomes, or it is posoccur, the phospholipid material tended to be sible that this material was in the processof evenly dispersed, the number increasing in being ingested. degenerating forms. During the period of logarithmic growth, l-4 small granules (up to The Pyronin-Methyl Green (PMG) Reaction 1 u in diameter) stained but after this period The above test was of interest since no these increased in size and number (up to 8) aggregations of RNA positive material were to approximately the size of the large toluidine noted until the second day for either species, blue and lipid staining granules. though the cytoplasm always stained positively. These staining aggregations were up Naphthol AS-BI Phosphate Method to 0.5 p in diameter, smaller, and fewer in All trypanosomespossessedone large body number than the large lipid or toluidine blue (about 2-3 p in diameter) which stained positive bodies (orthochromatic or metachro- positively for acid phosphatase. In some inmatic), and the organisms always contained stancesseveral bodies of varying sizes stained a number of non-staining vacuoles which cor- positively in each trypanosome, especially in responded in size with either the large lipid the rounded forms. There was little variation positive bodies or large metachromatic bodies in this pattern until the tenth day when there seen after toluidine blue staining. Pyronino- was a marked increase in positively staining philic bodies persisted in both species until inclusions. The differences in staining of inthe ninth day when the organismslost much dividual organismsprobably points to physioof their pyroninophilia, vacuolation being logical changes within the granules and the evident. The pyroninophilia was removed trypanosome body. The single large body from these organismsby ribonucleaseenzyme. demonstrated by this technique could well be the golgi apparatus. The smaller, more numerstaining bodies probably correspond with those which react positively for acid phosphaLipid was never absent from any organism. tase by the Gomori (1950) technique (Gurr, On Day 1, the lipid inclusions were small in 1958). SudaJt Black Reaction

B in Propylene

Glycol

ous

CYTOPLASMIC

INCLUSIONS

The histochemical examinations of the cytoplasmic inclusions and organelles other than those described earlier are summarized in Table I. The results obtained from this and the previous section have not unequivocally demonstrated the presence of more than one type of cytoplasmic body, though it is thought highly likely that this is the case. It is also conceivable that one basic type of cytoplasmic body is present which varies in reactivity during the growth of the organisms in culture. Both phenomena may also occur. It is here that the photomicrographs are of help.

The electron microscope studies detail several structures which are not visible under the ordinary light microscope. The mitochondrial nature of the kinetoplast and the origin of the mitochondria from this structure is only seen in certain sections of T. theileri (Fig. 2). This condition appears to be true of all trypanosomesstudied so far (Vickerman, 1962). In somesections which are not shown here small vesicles are formed within the mitochondria and are probable evidence of degeneration. The Golgi apparatus is situated in between the kinetoplast and vesicular nucleus in both T. theileri and T. melophagium (Figs. 2, 3, 4 and S), someof the vesicles of this apparatus are comparatively large. Scattered more or lessirregularly about the cytoplasm are electron-densestructures which are frequently rod-shaped. These may be aggregatesof ribosomessimilar to those reported from Strigomonas olzchopelti by Newton and Horne (1957) or symbionts, which multiply by binary fission. These are less obvious in older trypanosomes. Numerous fat globules are also present, especially in aging trypanosomesthough not confined to those and these are interspersed with vacuolated or partially vacuolated structures of varying sizes which are probably lysosomes. The larger of these vacuoles is occasionally seento have a double membrane structure (Fig. 6) and electron-

AND

29

ORGANELLES

dense material within certain of them (Figs. 3 and 7) is reminiscent of the dense bodies described from cytolysosomes of metabolically active cells by Napolitano (1963). The endoplasmic reticulum is also seen in several sections of both trypanosomes (for example, Fig. 2). There is also a number of structures whose nature is unknown, these being present in young actively growing cells (Fig. 2 [x] ) , though they may be a stage in the development of the lysosome and they are in the near vacinity of the Golgi apparatus. The almost complete vacuolation of degenerate trypanosomes is seen (Fig. 7) : vacuoles may be interspersed with fat. DISCUSSION The electron microscope studies coupled with the histochemical tests outlined demonstrate the complex system of organelles (mitochondria, Golgi apparatus, endoplasmicreticulum, ribosomes,and lysosomes)and inclusions (lipid) in both T. theileri and T. melophagium. Even though this is so this study does not leave one certain that the structures seen in electron photomicrographs can be accurately matched with the chemical reactivities seen in histochemical tests as outlined here. However analogy with studies on vertebrate cells permit a number of speculations which the writer feels may have some basisin fact. The electron photomicrographs and histochemical studies show the increased granulation or globule formation of trypanosomes as the medium in which they are growing ceases to provide a favorable environment, this being due in some measure to accumulating toxic metabolic products and the aging of individual cells. This is true of cultured forms of both T. theileri and T. melophagium, though demise of cultures is always more rapid in the case of the larger T. theileri. Electron photomicrographs of course demonstrate granule or globule formation which is not visible under the ordinary light microscope. It seemsprobably from the evidence pre-

30

HERBERT

Histochemical

Tests

Organelle or enzyme system Mitochondria

Test Metzner’s (Metzner Krause,

OIZ Trypanosoma

and

method and 1928)

Hirschler’s Janus

reference

method

green

Cytochrome oxidase

G.-Nadi Moog)

Succinic dehydrogenase

Blue tetrazolium (Gurr, 1958)

Alkaline inorganic pyrophosphatase

Acid inorganic pyrophosphatase

(1927)

fl

reaction (after of Pearse (1960) method

TABLE I theileri

and

Fixative

Trypanosoma

(if any)

melophagiuma Result (Refers to cytoplasmic structures cnly)

Altmann

Regarded as negative. A few granules were positive in a few trypanosomes seen better in T. theileri than T. melophagium.

Altmann

Negative

Supravital

Negative. The kinetoplast and numerous cytoplasmic bodies stained intensely green but no color changes were observed. Negative

None 1. Unfixed fresh and post fixed in form01 calcium.

Negative

2. 5 per cent saline

Negative

form01

Triphenyl tetrazolium chloride (Guha, Pyne, and Sen, 1956)

Supravital

Negative

Nitro-blue tetrazolium (after Machlas et al., 1957) method of Pearse ( 1960)

Supravital

Postive. The staining appeared in granules of 2-3~ in size, especially in rounded trypanosomes. Seen better in T. theileri than in T. melophagium.

Method (after Kurata and Maeda, 1956) of Pearse (1960)

1. Unfixed

Negative

2. Cold (4°C) form01 saline

Negative

1. Unfixed 2. Cold (4°C) form01 saline

Numerous ules

Method (after Kurata and Maeda, 1956) of Pearse (1960)

positive

gran-

Negative Lipase

Gomori’s method

(1952)

Tween

Esterase

Alpha naphthyl acetate method (Pearse, 1960) Dye used: Brentamine Fast black B salt

Cold (4°C) calcium

formol

Variable numbers tive granules

Cold

Acetone

Variable numbers of positive granules corresponding in number to those seen in the test for Lipase.

(4°C)

of posi-

CYTOPLASMIC

INCLUSIONS TABLE

Organelle or enzyme system

p glucuronidase

Test

and

AND

31

ORGANELLES

I (Continued)

reference

Fixative

(if any)

Alpha naphthyl acetate Diazo-red RC method (Gurr, 1958)

Cold (4°C) calcium

Ferric hydroxyquinoline method (Fishman Baker) of Pearse

1. Unfixed and (1960)

Result (Refers to cytoplasmic structures only)

form01

2. Cold (4°C) chlorol formalin

Few and smaller than the above

granules

Positive in T. theileri; a few positively staining bodies in a few T. melophagium

3. Cold (4”,C) form01 calcium

Negative

Cold (4°C) calcium

form01

Negative

a-Naphthyl phosphatase (after Gomori, 1951). Dye used: Naphthanil diazo blue i3 (Gurr, 1958)

Cold

Acetone

Negative

Calcium cobalt method (after Gomori, 1952) of Pearse (1960)

1. Absolute alcohol and ether 2. Form01 calcium 3. Ether

Negative

Lead phosphate method (Gomori, 1950) of Gurr (1958)

1. Cold (4°C) Acetone 2. Cold (4°C) formal calcium

Numerous bodies positive. Not the large lipid positive globules

Modified lead nitrate method (after Takenchi and Tanoue) of Pearse (1960)

Cold

(4°C)

Acetone

Numerous bodies positive. Not the large lipid positive globules

Naphthol-AS-B1 phosphate method (after Burstone) of Pearse (1960)

Cold

(4°C)

Acetone

Sometimes one large sometimes ule, globules

granmany

Calcium method (after Padykula and Herman) of Pearse (1960)

Unfixed

A few small (OS-1~) tive granules, not trypanosomes

posiin all

Lead nitrate method (after Wachstein and Meisel) of Pearse (1960)

1. Form01

Lipofuscins

Chrome alum hematoxylin method (after Gomori, 1941) of Pearse (1960)

Form01

Phagosomes

Strauss’ horseradish peroxidase-benzidine method (1961)”

Unfixed-post-fixed in form01 calcium

8-Hydroxyquinoline coupling axe dye (Pearse, 1960) Alkaline phosphatase

Acid phosphatase

Adenosine triphosphatase

a Where the results are identical in both b L. Light and Co. Ltd., England.

method

trypanosomes,

(4°C)

calcium

2. Unfixed calcium

no distinction

is cited.

A few small (OS--1~) positive granules, not in all trypanosomes I 2-6 granules were positive. More in T. theileri than in T. melophagium A

variable number of large positive granules in T. theileri, a few in T. melophagium

32

HERBERT

FIG. 2. Electron photomicrograph of in vitro cultured Trypanosoma the&&. Section of organism taken from a 4-day-old culture. ~39,000. The nucleus (N) is vesicular (n is nucleolus), and the well developed Golgi apparatus (G) is situated between it and the kinetoplast (K). The kinetoplast (K) is shown giving rise to the mitochondrion (M). It is probable that the mitochondrion (M?) is extensive at the opposite end of the organism as seen from the various sections of this body. The endoplasmic reticulum (E.R.) is well developed. Some small amount of lipid (L) is also present. Small vesicles (V) are present in the vicinity of the Golgi apparatus. Another body (X) of unknown nature is also present in this region. The structures (R) are possibly ribosomal aggregates. The large body marked Ly may be lysosomal in nature containing a number of cell bodies, one of which has a double membrane.

FIG. 3. Electron photomicrograph of in vitro cultured T. melophagium. Section of an organism taken from a 9.day-old culture. X48,000. The Golgi apparatus (G) is particularly well developed in this organism. Large vacuoles (Ly) which are thought to be lysosomes, some of which contain stellate material (p) that may be an accumulation of pigment or inorganic material. Some electron-dense material is also seen in this section which may be aggregates of ribosomes (R). The presence of electron-dense elongate material (H.P.?) may be a hyperparasite. For key to other structures see Fig. 2.

CYTOPLASMIC

FIG.

X48,000. veloped

4.

INCLUSIONS

AND

ORGANELLES

33

Electron photomicrograph of in vitro cultured T. theileri taken from a 4-day-old culture. This section shows the origin of the flagellum (F) and the reservoir (R) also the well-deGolgi apparatus. Small vesicle formation (V) is particularly evident in this region (see also Fig. 5).

FIG. 5. Electron photomicrograph of in vitro cultured T. the&& taken from a 4-day-old culture. X24,000. Numerous rod-shaped electron-dense material (H.P.?) is present, just as it was in T. nzelophagium (see Fig. 3). These may he aggregates of ribosomes, or hyperparasites. A structure with a question mark is unknown but may be a developing lysosome. L = lipid.

34

HERBERT

photomicrograph of in vitro cultured T. melophagium taken from a nine-day-old FIG. 6. Electron ture (X 120,000). This section shows the large lysosome body (Ly) with a clearly defined double membrane. The tron dense material is probably aggregated ribosomes. M is mitochondrion.

sented here and elsewhere (Herbert, 1965) that more than one type of cytoplasmic body is present in the trypanosomesstudied or, less likely, that the different sized bodies with different chemical affinities are but stagesin the development of one structure. Certain of the bodies which are seen in the cytoplasm are organelles while some are inclusions, and on this basis, structures previously referred to collectively as volutin require further characterization. Comparison of sizes of the cytoplasmic bodies as seen in the histochemical studies are not valid since the use of different fixatives and staining reactions induce enormous variation in the size of the organism and cytoplasmic bodies under consideration. The relative sizes of the cytoplasmic bodies, etc. are given, however, to permit comparison of results obtained from studies on other organisms. Ormerod (1961) has drawn attention to the impermanent nature of the refractile bodies (or volutin) which appear in the course of

culelec-

development of trypanosomes.An attempt has been made earlier (Herbert, 1965) to show that these so-called ‘volutin’ bodies of T. theileri and T. melophagium are impermanent structures and that this term appearsto encompass cytoplasmic bodies of varying size, state of development, and possibly function. Volutin has been described in various plants, fungi, bacteria, and the trypanosomidae and it has been common practice to refer to all bodies that induce metachromasia with metachromatic dyes as volutin. Further, a number of workers has also demonstrated RNA positive granules in trypanosomidae and have assumed that these are responsible for inducing the metachromasia.It is clear that this is not so, at least, in T. theileri and T. melophagium. The results of toluidine blue staining described here show that the number of bodies that stain metachromatically (i.e., volutin) increase as the organism ages and that even when these samebodiesdo contain ribonucleic acid (RNA) in quantities demonstrable by

CYTOPLASMIC

INCLUSIONS

AND

ORGANELLES

35

FIG. 7. Electron photomicrograph of in vitro cultured T. theileri taken from a 4-day-old culture. X48,000. This is a degenerating trypanosome and in conditions of healthy culture the organism is not full of very large vacuoles of Iipid (though some lipid is present) but a mass of fairIy well-defined bodies that are probably cytolysosomes (Ly). They are very similar to those described by Behnke (1963). For key to other structures, see earlier figures.

staining techniques, it seems that this is not responsible for inducing the metachromasia under the conditions of the test performed here. Preliminary observations had shown that formaldehyde-containing fixatives always permitted the best demonstration of metachromatic granules in trypanosomes. While prolonged fixation of the organisms in calcium form01 does cause the eventual loss of metachromasia, it is thought that the irregularity or impermanence of the metachromasia after toluidine blue-staining is more probably attributed to the instability of the polyphosphate content of these structures. Furthermore, ex-

traction of RNA with ribonuclease enzyme removes only the orthochromatic bodies. It is very rare for all trypanosomes from a culture to be without these metachromatically staining bodies, though one sees individual organisms without them, especially when the culture as a whole is commencing logarithmic growth. The lipid inclusions markedly increase in number and size with age as seen from Sudan black B staining, and no organisms have been seen free of them. Some of these larger inclusions at least correspond in size with those that react positively for phospholipid.

36

HERBERT

These correspond in size and number with those metachromatic bodies described above, though as shown previously (Herbert, 1965) pyridine extraction of lipid did not remove the metachromasia. It is therefore a matter of speculation at present whether these two chemical constituents are part of the same cell structure, though electron microscope studies demonstrate separate lipid and vacuolated bodies, and it is considered that the latter correspond with the metachromatic bodies visible after toluidine blue staining. Their instability and the demonstration here of acid phosphataseenzyme adds weight to the contention that these bodies contain polyphosphate though it is realized, that metachromasia can be due to numerous other chemical groupings besides high molecular weight phosphates. Cytochemical techniques involved in the demonstration of mitochondria proved to be unsatisfactory. The later demonstration of mitochondria (or possibly sections through one large mitochondrion which extends from the kinetoplast since studies have not been sufficiently extensive to determine this point) by electron photomicrographs made it necessary to have a fresh look at the cytochemical techniques. It was not possible,however, with the techniquesused to demonstrate mitochondria or mitochondrial enzyme systems,except by supravital staining in nitro-blue tetrazolium (Pearse, 1960). The discrete positively staining bodies were particularly numerous in rounded trypanosomes; they did not appear to form a continuous tube originating from the kinetoplast, though the latter always stained strongly. This finding is consistent with much of the work on supravital staining of trypanosomes and revolves round the ability of the dye to penetrate the trypanosomecell. It has been noted elsewhere (Herbert, 1965) that it is the rounded atypical trypanosomes which take up the supravital stain quickest and these survive for a shorter period in vitro than organismswhich do not take the stain. It has already been stated that

survives much longer in culture than does T. theileri, and as one might expect it does not take supravital stains into its body so quickly. This could account for the fact that the nitro-blue tetrazolium reaction did not work well as a stain for the mitochondria of T. melophagium. We do not fully understand the factors involved whereby certain trypanosomidae take dyes, drugs, or nutrients into their bodies either in vitro or in Go, while others in the sameenvironment do not. It does seem, however, from the literature that those trypanosomeswhich most readily assimilatedyes and toxic drugs supravitally are also those which are killed most quickly if the organisms are not removed from the antagonistic environment. Electron photomicrographs revealed small vesicle formation in the mitochondria of degenerating T. theileri and T. melophagium, and this may at least be part of the source of great accumulation of lipid in these organisms. It also seemspossiblethat much of the lipid seen in histochemical tests is protein-bound phospholipid (see Herbert, 1965), since phospholipid could only be demonstrated after the organisms were fixed in Lewitsky or Flemming fluid fixatives which, it is suggested, dissociated the phospholipid from the protein and rendered it demonstrable. Some of the lipid demonstrated by the Sudan black B method outlined was probably phospholipid associated with the mitochondrion, phospholipid micelles being associated with the structure and catalytic activities of such a structure in other cells. It is also possible that the phospholipid could also be associated with maintenance of the lysosomal membranes,since Holt (1959) has suggested that loss of phospholipid from a cell during fixation may also permit the lossof lysosomal enzymes. Adenosine triphosphatase enzymes are also associatedwith mitochondria and their histochemical demonstration by the tests recorded here is open to criticism. All that can be said is that trypanosomes possesspositively reT. melophagium

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INCLUSIONS

acting granules in much the same way described above in the demonstration of mitochondria, and one would certainly expect ATP to be present within mitochondria to act as a substrate. Furthermore, the presence of inorganic phosphatehas been demonstrated from these trypanosomes, and its associationin the formation of ATP in vertebrate cells is well known (Green, 1964). Specific reactions for organic and inorganic alkaline phosphatase enzymes, however, have resulted in failure. Experiments on the in vitro culture of trypanosomes had demonstrated that certain unspecified molecules which were unable to passthrough a semipermeablemembranewere required if the organismswere to live (Herbert, 1963). Just how the organism absorbs these molecules is a subject of considerable interest. The identification of a number of acid phosphatase and acid inorganic pyrophosphatase localizations by histochemical tests and neutral red-staining bodies after supravital staining is of interest in this context, and the demonstration of phagosomes (= digestive lysosomes) offers a hypothesis as to how these macromolecules find their way into the trypanosome cell. Karnovsky (1962) has discussedthe importance of pinocytotic activity within mammalian cells. The demonstration of acid phosphatase enzyme activity is associatedwith an active membrane phenomenon in reticuloendothelial cells, kidney cells, polymorphonuclear leucocytes and wherever else these processesoccur. Various macromolecular substances stimulate these processesso that the cell forms vesicles containing ingested material, somesubstancesin the environment (e.g., gamma globulins) stimulate the processmore than others, while some other substances antagonize it. During the time of this membrane activity it is possible that metabolic inhibitors leak into cells, whereas they can not do so at other times. It is further thought that phosphataseenzymes are associatedwith the detoxification of such inhibitors and that cells with granules containing acid phospha-

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tase enzymes have enhanced ability to deal with inhibitors over those cells that have no such granules. After activation these granules, which are lysosomes,are larger and more numerous. It is thought conceivable that the lysosomesof T. theileri and T. melophagium may be capable of all the mentioned functions that lysosomesof vertebrate cells are capable of and that this may be a widespread phenomenon in all trypanosomidae, and perhaps in other protozoa as well. Seaman (1961) has demonstrated the formation of pinocytotic vesiclesin Tetrahymena sp., Holter (1959) in Chaos chaos, and Steinert and Novikoff (1960) in Trypanosoma mega; and acid phosphatase activity has been demonstrated in Trichonympha (Grimstone, 1959), Para.me&m mdtimicronucleatum (Muller and T&o, 1962), T. rhodesiense, and T. mega (Brooker and Vickerman, 1964). Other records exist for other protozoa. The actual formation of pinocytotic vesicles has not been demonstrated in T. theileri and T. melophagium though structures are seen in photomicrographs which are bounded by a unit membrane and these are probably responsible for the acid phosphatase activity. The association of these two phenomena fulfills, according to Novikoff ( 1961)) the concept of the lysosome. Many of these small vesicles are in the vicinity of the Golgi apparatus which in turn is also near the reservoir at the base of the flagellum. It is conceivable that pinocytotic vesicles may develop at this site though the aggregation of granules (as seen under the ordinary light microscope) early on in cultural growth posterior to the nucleus has led the writer to wonder whether pinocytosis was occurring in that region. de Duve (1962) has pointed out that it is necessary to distinguish different types of lysosomes in vertebrate cells, i.e., ‘storage granules,’ ‘digestive vacuoles,’ ‘residual bodies,’ and ‘autophagic vacuoles,’ or ‘cytolysosomes,’ the first three being directly involved in digestion and the latter with

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degradation of cellular structures. According to de Duve (l.c.) the association of the ribosomes and endoplasmic reticulum with the phagosome ultimately leads to the formation of digestive vacuoles, the residual material being ultimately eliminated from the body. Phagosomes are present in both T. theileri and T. melophagium as demonstrated by the method of Strauss (1961) and a few granules stain positively for lipofuscins in a few trypanosomes; the lipofuscins of vertebrate cells being described as part of the lysosomal residual body. The lysosomal residual body is probably derived from both phagosomesand the autotrophic vacuoles. If one postulates that a similar situation holds for the trypanosomes described here, one can explain the fleeting appearanceof bodies such as ribosomes (or ribosomal aggregates), such as were seen during histochemical investigations. Phagosomes, or digestive lysosomes, are concerned in the feeding process of cells and by virtue of their acid hydrolytic enzyme systems, with detoxification of material of endogenousor exogenoussource, (Karnovsky, 1962). If these remarks are confined to materials of exogenous source we can see that pinocytosis or phagocytosis by phagosomes may permit the entry of undesirablemolecules which could not enter when the organism is not undergoing pinocytosis. The supravital staining of trypanosomes in vitro is only possible at certain times, and if one accepts the above view this presumably occurs when the organisms are undergoing pinocytosis, those that assimilatethe toxic dye first are the first to be killed. It is therefore, not difficult to imagine that if a toxin is imbibed in large quantity the lysosomemay be unable to complete detoxification so that the cell is killed. This it is thought could be what happens when a culture declines in vitro or when drugs or antibodies affect trypanosomes in viva. Individual cells that are degenerating in an otherwise equable environment prob-

ably undergo autolysis by the agency of cytolysosomes.Attempts to demonstrate other enzyme systems in lysosomes besides acid phosphatase has met with some difficulties. It has been possibleto demonstrate a positive result for esteraseand p glucuronidase,though results were not always consistent. Trypanosoma theileri gives more satisfactory positive results in this context. Herein lies the difficulty of histochemical studies, that certain tests may sometimeswork, but not always, depending upon the physiological conditions of the trypanosome under study (particularly in relation to their feeding) and the ability of the dye to gain access. It seemsreasonable to suppose that the organelles within the trypanosome are in a dynamic state, their condition varying with the state of the organismsin which they are found, this in turn being dependent upon the environment which is external to the trypanosome. The demonstration of an extensive Golgi apparatus in trypanosomes, which are in a healthy state of growth, is evidence of an active metabolism, this state being absent from old culture forms. The functional continuity of membrane-bound organellesis emphasized by Novikoff et al. (1963) from work on vertebrate tissue, there being a turnover of cell membranes during phagocytosis or pinocytosis. According to these workers there is a development of the endoplasmic reticulum from the nuclear membrane and a reorientation and transformation of the endoplasmic reticulum to produce the Golgi cisternae. The association of the Golgi with the acid phosphataserich lysosomesremains unproved but offers a working hypothesis in explaining the function of these organelles. It doesnot seemunreasonableto supposethat this basic pattern of events holds true for trypanosomes and other protozoa. There may be somedifferencesin the mechanismof events to accommodate the specific mode of life of the organismsconcerned.

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INCLUSIONS

I am indebted to Dr. R. G. Bird of the London School of Tropical Medicine and Hygiene for preparing the trypanosomes for electron photomicrographs and to Dr. N. W. Runham for advice on histochemical techniques. REFERENCES BAKER, J. R. 1946. The histochemical recognition of lipase. Quarterly Journal of Microscopical Sciences 87, 441-470. BEHNKE, 0. 1963. Demonstration of acid phosphatase-containing granules and cytoplasmic bodies in the epithelium of foetal rat duodenum during certain stages of differentiation. Journal of Cell Biology 18, 251-265. BROOKER, B. E., AND VICKERMAN, K. 1964. Acid phosphatase in trypanosomes. Transactions of the Royal Society of Tropical Medicine and Hygiene 58, 293-294. DUVE, C. DE. 1962. The lysosome. Scientific American 208 (S), 62-72. GOMORI, G. 1952. “Microscopic Histochemistry.” Univ. of Chicago Press, Chicago, Illinois. GREEN, D. E. 1964. The mitochondrion. Scientific American 210, 63-74. GRIMSTONE, A. V. 1959. Cytoplasmic membranes and the nuclear membrane or the flagellate Trichonympha. Journal of Biophysical and Biochemical Cytology 6, 369-382. GUHA, A., PYNE, C. K., AND SEN, B. B. 1956. Cytochemical studies of mitochondria in leptomonad forms of Leishmania donovani, the Kala azar parasite. Journal of Histochemistry and Cytochemistry 4, 212-216. GURR, E. 1958. “Methods of Analytical Histology and Histochemistry.” Leonard Hill, London, HERBERT, I. V. 1961. Bovine trypanosomiasis due to Trypanosoma tkeileri Laveran, 1902 and its occurence in Eire. Irish Veterinary Journal 15, 230-236. HERBERT, I. V. 1963. Laboratory studies on a Trypanosoma (T. theileri, Laveran) of Cattle in Britain. Ph.D. Thesis. University of Nottingham, England. HERBERT, I. V. 1965. Cytochemistry of in vitro cultured Trypanosoma theileri. Experimental Parasitology 16, 348-362. HIRSHLER, J. 1927. Uberein einfaches Vorgehen zur Darstellung des Golgi-apparatus und der Mitochondrien bei Wirbellosen Zeitschrift fiir Wissenschaftliche Mikroskopie und fiir Mikroskopische Technik 44, 216-218. HOLT, S. J. 1959. Factors governing the validity of

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K. 1962. A mechanism of cyclical development in trypanosomes of the Trypanosoma brucei subgroup: a hypothesis based on ultrastructural observations. Transaclions of the Royal Society of Tropical Medicine and Hygiene 56, 487-495. WENYON, C. M. 1926. “Protozoology,” Vols. I and II. Bailliere, Tindall and Cox, London.

VICKERMAN,