COMPARATIVE ULTRASTRUCTURAL STUDY BETWEEN YOUNG

AND ADULT FORMS OP TRYPANOSOMA LEWISI

APPROVED»

It Ma j o^Profess or

Qfcgsu ii. Q J U Minor Professor

\ \J r _ _

Director of the Department of Biology

Dean of the Graduate School <

iV

Abdul-Salam, Jasem VI., Comparative Ultras true tural

Study Between Young and Adult Forms of Trypanosoma Lewisi.

Master of Science (Biology), August, 1973> 57 pp., 20 illus-

trations, bibliography, 51 titles.

The purpose of this study was to examine the ultra-

structure of both young (dividing form) and adult (stationary

form) of Trypanosoma lewisi. Changes observed between the

two forms of the organism were related directly to data ob-

tained from previous comparative biochemical studies conducted

on these forms of Trypanosoma lewisi. The overall subcellular

arrangement of both young and adult forms shares many char-

acteristics with other members of the genus Trypanosoma

which already have been studied. Definite changes in the

size of the nucleolus, mitochondria, arrangement of the

granular reticulum and Golgi complex were observed between

the two forms. The presence of a large nucleolus, vesicular

Golgi complex, conspicuous granular reticulum, and abundance

of smooth-walled vesicles throughout the cytoplasm of young

trypar.osorr.es indicated high secretory activity at this stage

of growth. Existence of long mitochondria in adult trypan-

osomes was a characteristic feature absent in young

trypanosomes. High respiratory quotients reported in adult

trypanosomes could be associated with the presence of large

and highly developed mitochondria. A marked increase in

the number of inclusion bodies located in the posterior end

of adult trypanosoraes was observed. The nature of these

bodies is still obscure. Previous ultrastruetural studies

of flagellae of several members of the genus Trypanosoma

indicated termination of central tubulars of the axoneme

in the kinetosomal region. This study clearly showed the

penetration of central tubulars of the axoneme deep in the

cytoplasmic matrix, surpassing both kinetosomal plates.

COMPARATIVE ULTRA5TRUCTURAL STUDY BETWEEN YOUNG

AND ADULT FORMS OF TRYPANOSOMA LEWISI

THESIS

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillments of the Requirements

For the Degree of

MASTER OF SCIENCE

3y

Jasem M. Abdul-Salam, B.A.

Denton, Texas

August, 1973

TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS ii

INTRODUCTION ' 1

MATERIALS AND METHODS 9

RESULTS . 11

DISCUSSION 48

BIBLIOGRAPHY 53

LIST OP ILLUSTRATIONS

Figure Page

1.1 Light Microscopy, Trypanosoma lewisi, young form, Giernsa stained . . . . 19

1.2 Electron Photomicrograph, Trypanosoma lewisi, young form, overall view of the organism . 19

2.1 Light Microscopy, Trypanosoma lewisi, adult form, Giemsa stained . . . . . . 21

2.2 Electron Photomicrograph, Trypanosoma lewisi, overall view of the organism . . . . . . . 21

3. Electron Photomicrograph, Trypanosoma lewisi, young form, the anterior end of the organism . . . 23

4. Electron Photomicrograph. Trypanosoma lewisi, adult form, the anterior end of the" organism 23

5. Electron Photomicrograph, Trypanosoma lewisi, adult form, the flagellum and the sub-pellicular microtubular 25

6. Electron Photomicrograph, Trypanosoma lewisi, young form, flagellum-associated reticulum and sub-peilicular microtubulars 25

7. Electron Photomicrograph, Trypanosoma lewisi, young form, intra-flagellar structure . . 25

8. Electron Photomicrograph, Trypanosoma lewisi, young form, granular reticulum system ." . 27

9. Electron Photomicrograph, Trypanosoma lewisi, adult form, agranular reticulum system, sub-peilicular microtubulars, and mito-chondria 29

10. Electron Photomicrographt Trypanosoma lewisi, your^g form* bvsrall VIgV of organism . . . . 31

11. Electron Photomicrograph, Trypanosoma lewisi. adult form, agranular reticulum system with sac of secretion . . . . . . . . . . . 33

12. Electron Photomicrograph, Trypanosoma lewisi, . young form, anterior part of the organism during division 35

13. Electron Photomicrograph Trypanosoma lewisi, adult form, mitochondria and agranular reticulum . . . . . . . . . 37

14-. Electron Photomicrograph, Trypanosoma lewisi, young form, granular reticulum in the nucleus vicinity . . . . . . . . . 39

15. Electron Photomicrograph, Trypanosoma lewisi, adult form, posterior end of the organism. . 41

16. Electron Photomicrograph, Trypanosoma lewisi, adult form, mitochondria and "kinetoplast . . 43

17. Electron Photomicrograph, Trypanosoma lewisi, adult form, vacuole surrounded by agran-ular reticulum . . . . . . ^3

18. Electron Photomicrography, Trypanosoma lewisi, adult form, posterior end of the organism. . kj

19• Electron Photomicrograph, Trypanosoma lewisi, young form, posterior end of the organism. . 45

20. Electron Photomicrograph, Trypanosoma lewisi, young form, high magnification of the kinetoplast and flagellum emergence from the cell . 47

INTRODUCTION

Trypanosoma lewisi is a typical member of the genus

Trypanosoma. The name of the genus was proposed by Gruby

•in 18^3. During the long history of this genus there have

been many attempts to subdivide it into subgenera or create

new genera. This conflict in classification was the result

of the discovery of new species and development of new

methods in the determination of species. Attempts to add

new genera did not succeed because of the great similarity

in the morphology of all species under study, although they

were host-specific parasites. Pleomorphism throughout the

life cycle of these parasites introduced another obstacle

for taxonomists.

After a great deal of controversy Hoare (20) initiated

a new classification scheme, which is recognized by most of

the workers with this group of parasites. The new classifi-

cation is based on the division of the genus Trypanosoma

into two major divisions or sections. Some of the criteria

upon which this classification is based are phylogenetic

consideration and conventional diagnostic characters such

as morphology, method of reproduction in the vertebrate host,

the position and size of the kinetoplast, and biochemical

data. Section A, or Stercoraria, the name of this section

indicates the development of the organism in the vector is

completed in the posterior parte cf the digestive system, and

transmission is accomplished by fecal contamination. The

Stercoraria are divided into four subgenera r Metatrypanum,

Schizotrypanum, Endotrypanum, and Herpetosom. Trypanosoma

lewisi is classified under the subgenus Herpetosom, Morpho-

logical characteristics shared by the Sterocoraria are -

(1) presence of free flagellumj (2) large and not terminal

kinetoplast j (3) posterior end of the body is pointed. Sec-

tion B or Salivaria also is divided into four subgenera t

Dultonella, Nannomonas, Pycnomonos, and Trypanzoon, which

originally included "lewisi." The development of Salivaria

members is completed in the anterior parts of the vector's

digestive system. The parasite is transmitted to the host

by direct inoculation. Species under the Salivaria section

share these morphological characteristics t (1) free flagellura

not always present? (2) the kinetoplast is terminal or sub-

terminal; (3) the posterior end of the body is usually blunt.

It is important to mention that most of the pathogenic try-

panosomes are placed under the Salivaria section, such as

the famous African species, Trypanosoma gambiense and Trypan-

osoma rhodesiense.

Because of the cosmopolitan distribution of Trypanosoma

lewisi and its relative ease of maintenance in the laboratory,

it has teen the subject of numerous studies all over the

world, and especially in the United States. The natural life

cycle of Trypanoscnga lewis! is divided between '/..any species

of wild rats of the genus Rattus as the vertebrate host and

the common rat flea Fasciatus nosophyllus as the invertebrate

vector. The rat acquires the infection by ingesting contam-

inated. flea feces. After several hours the parasite appears

in the circulatory system as an extracellular parasite. The

first week of the infection marks a rapid reproduction by

longitudinal and multiple fission. Reproduction terminates

after the first week, and the growth curve reaches a plateau

for three days before the number of organisms decreases

sharply. It takes from ten days to several months for organ-

isms to disappear completely from the blood, leaving the host

immune to another infection. The second cycle of reproduc-

tion lasts about fiye days in the vector's digestive system.

The flea acquires the infection by feeding on the blood of

an infected rat. Within the epitheliae cells lining the

stomach of the vector, the parasite is transformed from the

trypanosomal stage to the crithidial stage during the repro-

duction. Crithidial forms migrate to the rectum of the flea,

where they become attached to the highly folded tissues. In

the rectum the crithidia transforms again to the trypanosomal

form, while undergoing longitudinal fission. The infective

forms, which are better known as metacyclic forms, are passed

out with the flea's feces. The parasite life cycle is

5; Hr'

continued if the contaminated fecal materials are ingested

by the vertebrate host.

As a typical member of the genus Trypanosoma, Trypanosoma

lewisi share the following morphological features with other

members of the genusi (1) a body flattened and elongated in

the trypanosomal stage and round in the crithidial stage?

(2) the nucleus is round or oval, found in the anterior part

of the body in the trypanosomal stage and in the middle of

the body in the crithidial stage? (3) a spherical, rod, or

disc-shaped kinetoplast located posterior to the nucleus in

the trypanosomal stage and anterior to the nucleus in the

crithidial stage; (^) a flageHum arising from the blepharo-

plast, which is located anterior to the kinetoplast? (5) in

the trypanosomal stage the flagellum runs along the border

of the cell membrane, forming an undulating membrane, and a

free anterior flagellum. Morphologically there are two dis-

tinct forms of the parasite in the blood stream of the host.

The young or dividing form is found from the first day of the

infection to the sixth day, while the adult form or stationary

form is found from the seventh day until the end of the in-

fection (^2). The young trypanosomes exhibit a variety of

sizes ranging from 10 to U0 microns, and are usually fat and

spindle-shaped. The adult forms are uniform in size 28 to

^0 microns, and are long and slender in shape.

In 192^, Taliaferro (42) described the immunological

reaction of a rat infevIwith Tv-;yT^nosoma lewj.si* A series

of immunological studies was conducted by the same author

( 3» -6), elucidating factors involved .in the mech-

anism and naming the reproduction-inhibitor ablastin. .Recent

immunological investigations by D'Alesandro (8, 9) indicate •

the presence of three separate stages associated with spe-

cific factors that help in the defense mechanism. First, a

reproduction-inhibiting antibody (ablastin)r second, a try-

panocidal antibody detected during the first crisis? and

third, a second trypanocidal antibody which terminates the

infection. Changes in the antigenic structures were divided

between the young and adult forms of the parasite (11, 12).

Numerous studies have been published on the biochemical

activity and composition of Trypanosoma lewisi throughout the

infection. Moulder (19^8), Ryley (1951)» Thurston (1938), and

Lincicome and Warsi (1966) are some of the pioneer investi-

gators in this area. Recently, Hibbard and Dusanic (18, 19)

conducted a very extensive study on the metabolism of Trypan-

osoma lewisi, conforming to most of the data obtained by the

previous investigators. Oxygen utilization was found to be O

52 aI and 100 jil Og consumed/10 trypanosomes per hour for

young and adult forms, respectively. The respiration quo-

tients were found to be 0.96 and 1.01 for young and adult

forms, respectively. The above data show 00/glucose ratios

of young trypanosomes significantly lower than the adult

forms. A comparative study between mitochondrial enzymes

of young and adult.forms of Trypanosoma lewisi (13) indi-

cates a significant difference in the level of isocitrate

dehydrogenases and ATP synthesizing systems.

Talioferro and Piz,zi, in i960 (^7), demonstrated the

rate of protein synthesis in Trypanosoma lewisi, Trypano-

soma equiperdum. and Trypanosoma cruzi, using labeled

precursors. The results indicate a high rate of incorpora-

tion by young forms of Trypanosoma lewisi, while the rate

is lower in the adult form. Amino acids analysis of

different species of trypanosomes by Williamson and Desowtiz

(1961) indicated a similarity between amino acids found in

these organisms, and other protein such as bovine albumin.

Data from the same experiment showed that Trypanosoma lewisi

contained only 0. -3 percent free amino acid while other

trypanosomes contained 2-3 percent free amino acid. Finally,

a quantitative and qualitative study on Trypanosoma lewisi

biochemical composition was conducted by Hibbard and Dusanic

(19)• Total amounts of protein for young and adult forms

were found to be 23.0 XIO"^ and 85.3 XIO"" g protein per

trypanosomes. The authors explained that the increase in the

percentage of protein per unit of dry weight during the in-

fection was due to decrease in the total dry weight of the

trypanosome rather than increase in protein synthesis. RNA

content was shown to be 12.59 X10 pg per young trypanosome,

-7

and 2.53 X10 p.g per adu.lt trypanosome. The above data in-

dicate a decrease in nucleic acid and protein synthesis in

the adult form. Similar results were obtained in biochemical

studies conducted on Trypanosoma lewisi (14).

Recently, electron microscopy has been utilized to reveal

fine structures of various members of Trypanosomatidae, Try-

panosoma cruzi, Trypanosoma lewisi. Trypanosoma equiperdum.

Trypanosoma conorrhini, Trypanosoma mega, Trypanosoma congo-

lense, Trypanosoma brucei, Trypanosoma gambiense, Trypanosoma

raiae, and Trypanosoma avium are some of the Trypanosomatidae

which have been subjects of numerous ultrastrueture studies

(1, 2, 3, 21, 29, 30, 35, 37, 38, 39, 40, 41, 49, 50, 51).

The study of the whole-mount specimens of Trypanosoma lewisi

was conducted by Kleinschmidt and Kinder in 1950 (22), just

a few years after the invention of this technique. In 1964,

Judge and Anderson published a brief study on the ultrastrue-

ture of thin sections of adult forms of Trypanosoma lewisi

(21). Later, in 19^5, Anderson and Ellis published a more

detailed study oh the adult form> devoted extensively to the

structure of the flags Hum, microtubules, and the kinetoplast

(2 ) .

The objective of this study is to investigate the ultra-

structure of young and adult forms of Trypanosoma lewisi.

The comparative .investigations will be based on previous

8

physiological, biochemical, and immunological differences

"that have already bser; -nstfiDi,'!.b§'tween "the "two forms#

Emphasis will be placed on the shape and size of the nucleus

and the nucleolus, changes in the endoplasmic reticulum,

Golgi complex, and inclusion bodies.

MATERIALS AND METHODS

Trypanosoma lewisi was maintained in the laboratory by-

weekly intraperitonial injection of uninfected white rats

with three drops of blood from seven-days-infected rat mixed

with three ml of saline solution. Infected rats were bled

by cardiac puncture three and ten days after the infection

to collect young and adult forms of the parasite. Organisms

were separated from red blood cells by differential centrifu-

gation methods.

The blood was centrifuged at 500 r.p.m. for five minutes

to isolate the organisms in the supernatant. The supernatant

was centrifuged again at 3000 r.p.m. for 30 minutes at 4° C.

After the second centrifugation procedure the organisms were

concentrated in a white pellet. During the isolation pro-

cedure the organisms were kept in an ice-bath.

Preparation for electron microscopy studies starts with

fixing the organisms in cold 1.5 percent solution of osmium

tetraoxide in vernal acetate buffer (pH 7.^-7«5) for* thirty

minutes at 4° C (3*0 • The fixative was aspirated and the

organisms were washed with vernal acetate buffer. The buffer

was removed and organisms were dehydrated in an ethanol

series and infiltrated with propylene oxide before embedding

in Spone 812. Sections were cut with a Sorval MT-2

10

ultramicrotome using glass knives. Sections were collected

on copper grids, and stained for three minutes with uranyl

acetate and five minutes with lead citrate. Sections were

observed by RCA EMU-3& electron microscope.

RESULTS

Examination by light microscopy reveals the external

morphology of young and adult forms of Trypanosoma lewisi

(Figures 1.1 and 2.1). Both forms of the organism are

spindle-shaped with a dark nucleus in the middle of the body

and a kinetoplast located in the posterior end of the organ-

ism. The flagellum emerges from the posterior end of the

body forming an undulating membrane by adhering to the pel-

licle as it proceeds to the anterior part of the body. The

organism's movement in the blood of the host is achieved by

rapid vibration of the flagellum and undulating movement

along the body, under the light microscope young and adult

forms of Trypanosoma lewisi are easily characterized on the

basis of external morphological differences. Young trypano-

somes exhibit a variety of shapes and sizes, mostly broad,

ranging from 15 to 25 microns in length. On the other hand,

adult trypanosomes are elongate, slender and uniform in size,

approximately 28 microns long.

In general, the ultrastrueture of both forms of Trypano-

soma lewisi have many basic characteristics shared with all

other members of genus Trypanosoma which have been studied

so far. There are several structural differences observed

between the two forms of Trypanosoma lewisi which could be

11

12

attributed to the growth stage of the trypanosome and possible

changes evoked by the host defense mechanisms throughout the

infection course. Those differences observed between the

two forms of Trypanosoma lewisi will be discussed in some

detail as we proceed in surveying the general ultrastrueture

of the organism.

Pellicle.--A unit membrane consisting of three layers

of membranous structure covers both the body and the flagellum

of the trypanosome (Figures 5 and 6). In a cross-section the

three membranous layers of the pellicle are seen as two dark

layers, osmiophillic in nature, with a low density layer in

between. Thin osmiophillic fibers cover the pellicle surface

of the flagellum and the body (Figure 5). This surface coat

is not well defined as is the case in other species of trypan-

osome s (H-9, 50).

Sub-pellicular microtubules. -—Beneath the pellicle of

the trypanosome lie highly organized microtubules arranged

in parallel lines extending longitudinally along the entire

length of the body (Figures 1, 2, 5, 6, and 9). This is a

unique feature observed in all other hemaflageHates. The

resolution of the electron micrograph does not allow the

examination of micrctubular sub-units, which are known to be

made up of ten to twelve tubules (2). The exact function of

sub-pellicular microtubules is not known, although several

proposals were adapted, such as contraction, coordination,

13

and support of the organism. At the site of the attachment

of the flagellum to the pellicle iour microtubules form a

unique association with cytoplasmic granular reticulum (Fig-

ures 5 and 6). This specialized organelle has been found

also in Trypanosoma vivax, Trypanosoma cruzi, Trypanosoma

brucei, and Trypanosoma congolense (^8). It has been proposed

that granular reticulum connected to microtubules may serve

as channels for transporting protein to the outside (48).

Nucleus.— The nucleus is surrounded by the usual double-

membrane structure with pores (Figures 1.2, 2.2, 3» and 4).

The outer membranous envelope in young trypanosomes is con-

tinuous with granular reticulum (Figure 1*0 . The nucleolus

(karyosome) consisting of two masses of electron dense material

is located in the middle of the nucleus. One of the main

differences between the two forms of Trypanosoma lewis.i is

the size of the nucleolus. Young trypanosomes are distin-

guished easily from adult trypanosomes by the presence of

dense and larger nucleoli (Figure 1.2).

FlaReHum.—The flagellum emerges from the posterior

end of the trypanosome in a close proximity to the kineto-

plast. At the point of the emergence from the body a flagellum

pocket is formed by inward invagination of the pellicle (Fig-

ure 20). In a. cross section the flagellum's axoneme consists

of the usual nine peripheral pairs of tubulars and two cen-

tral single tubulars (Figures 1.2 and 2.2). Each of the nine

peripheral tubulars consists of two tubulars fused together

with l a t e r a l arms extending, out of theiii in a clocKwise direc-

tion (Figure 5)• Along t he flagellar axoneme the paraxial

rod is seen as a dense granular structure (Figure 5)• In a

longitudinal section t h e paraxial rod has a lattice-like

appearance formed by filaments running in different directions

(Figure 7)• The kinetosome is located at the base of the

flagellum facing t he anterior portion of the kinetoplast.

It is made of two kinetosomal plates, shown in the electron

micrograph as two dense lines perpendicular to the axoneme

(Figure 20).

There is no evidence for a direct contact between the

base of the flagellum and the kinetoplast (Figure 19)• How-

ever, a dense cytoplasmic matrix appears to serve as a bridge

between the base of the flagellum and the anterior membrane

of the kinetoplast.

Kinetoplast.—The kinetoplast consists of two main re-

gions enclosed within the same double membrane. The anterior

portion of the kinetoplast is made up of disc-shaped osmio-

phillic tangles of fibers oriented in a longitudinal direction

with rays of thin fibers radiating toward the posterior part

(20). The posterior region of the kinetoplast exhibits a

typical mitochondrial appearance with cristae embedded in

an amorphous matrix. Mitochondrial-like portions of the adult

trypanosome's kinetoplast are elongated, extending toward the

15

anterior part of the body (Figures 16 and 18), while they are

rounded in young tryparvnor^es locatcd in The posterior end

of the body (Figure 19).

Cytoplasmic membrane systems.—Granular and agranular

membranes varying in their osmiophillic property are found

in the cytoplasm of both adult and young Trypanosoma lewisi.

In the cytoplasmic matrix of young trypanosomes two prominent

granular reticulum systems are found. The first one is found

in the nuclear region while the other is found in the poster-

ior end of the body near the kinetoplast (Figures 3» 1^» 15»

and 19). The granular system consists of several cisternae

arranged in a concentric fashion. Under high magnification

smooth-walled vesicles are seen attached to distal ends of

cisternae, with more vesicles of the same nature found free

in cytoplasm (Figure 8). Vesicles appear to be formed be-

tween the granular reticulum cisternae before they are pinched

off at the distal ends. Adult's granular reticulum does not

show the highly specific compact arrangement found in young

trypanosomes. They are either dispersed throughout the cyto-

plasm or found arranged in two to four rows of cisternae in

the periphery of the body (Figures 2.2, 5» and 9). Several

dense granulars are observed in the cytoplasm of young and

adult trypanosomes (Figures 1.1, 2.1, 11, and 19). The nature

and function of these granulars have been the subjects of

several studies, but so far there are no convincing

16

interpretations. Ormerod associated the appearance of gran-

ulars in different speuies of trypanosomes with host cell

immune reaction (32, 33)•

Inclusion bodies.—Inclusion bodies are observed exclu-

sively in the posterior ends of both young and adult trypanosomes

(Figures 10, 15, and 18). The number of inclusion bodies is

higher in the adult trypanosomes. Inclusion bodies of the

same nature have been reported in the same region of several

other species of trypanosomes. Those bodies are believed to

be the by-product of metabolic activity.

Agranular reticulum system termination in sacs of secre-

tion are observed in both forms of Trypanosoma lewisi (Figures

1.2, 3» and 11). Vacuoles are observed only in the adult form

of Trypanosoma lewisi (Figure 17).

The Golgi apparatus of adult trypanosomes shows the

classical lamellar arrangement found in the posterior end of

the organism (Figure 2.2), whereas the Golgi apparatus of

young trypanosomes consists of aggregates of small vesicles

found in the middle of the organism (Figures 1.2 and 10).

Mitochondria.—'Adult trypanosomes possess a long tubular

mitochondrion attached to the kinetoplast by a narrow tubular

structure (Figures 1, 16, and 18). In some cases the mito-

chondrion extends to the nuclear region terminating in a

large spherical mitochondrion (Figure *0 . Young trypanosomes,

on the contrary, possess smaller mitochondria which are

17

spherical in shape, with lateral growth (Figures 12 and 19).

There is no indication of a physical connection between,

young trypanosomes' mitochondria and the kinetoplast. Both

young and adult trypanosomes* mitochondria are surrounded

by double membranes with cristae embedded in an amorphous

matrix which is easily distinguished from the cytoplasmic

matrix by the absence of ribosomes.

18

Figure 1.1 Smear of young form of Trypanosoma lewisi.

Stained with Giemsa, light microscopy. X 1,400.

Figure 1.2 Trypanosoma lewisi. young form. Longitudinal

section showing nucleus (N) with its dense nu-

cleolus (nu), nuclear membrane (nm), pellicle

(P), granular reticulum (gr), sub-pellicular

microtubules (pm), vesicular Golgi complex (G),

cross section of a flagellum (F), mitochondria

(M), and angular reticulum (ar) attached to sac

of secretion (sar). Several dense granules are

seen scattered throughout the cytoplasm (g).

X 30,000.

19

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20

Figure 2.1 Smear of adult form of Trypanosoma lewisi.

Stained with Giemsa, light microscopy. X 1,400.

Figure 2.2 Trypanosoma lewisi. adult form. Longitudinal

section showing the nucleus (n) with its

nucleolus (nu), nuclear membrane (nm), granular

reticulum (gr), elongated mitochondrion (M),

lamellar Golgi complex (G), pellicle (P), and

a cross section of a flagellum (F). X 42,000.

21

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22

Figure 3. Trypanosoma lewisi, young form. Longitudinal

section through the anterior end of the organism

showing granular reticulum (gr) and vesicular

Golgi complex (G) in the vicinity of the nucleus

(N). X 30,000.

Figure b. Trypanosoma lewisi, adult form. Longitudinal

section through the anterior end of the organism

showing a nucleus (N) with two nucleoli (nu) and

an elongated mitochondrion (M). X 22,000.

*- 'X .

f?-j-„- -r" '5

^ : f y

$ ' . * r , ' / ; >" J # ^ 7

ft /'„ *- -" - -

\ J 0 ^ - ' : 4 -

t ' ^*" *• » " "

s& y

' • •'•:" ' ' . € ; • .

v - % *V-,V- :>

;% ,

' ,-r-f'y. — ^ " ^ v

- • ; T |

; ; p Q i i § S

gg/i? a*-r ^ £"

» / - • . . ^ - V '

* 4 - &•<**

-- . - f v : - <r ^ z-, - y ' '

•*"*- J J ' i </--

1 > I V v / * ' - # • - '-4

'" - ' y t -

i >„<• - > - T . Isfe*.

5-''"V>rS&?

• 5 - v ^ % v 4 s - « *

m ^ - - •

~"4'^_ * f

- ? X \>*

V ' ' ' ' ^

\&>

: X

it-* •1:): -n

a^ te4>%Sy>is J^ fe , - % £ ^ k J £ ' '

' -l. * - ''1% vi - -Y '

2k

Figure 5- Trypanosoma lewisi, adult form. Oblique section

demonstrating clearly the "9 + 2" pattern of the

axoneme (ax), paraxial rod (pr), and lateral arms

(la) extending from the nine peripheral tubulars

(pt) in a clockwise direction. The sub-pellicular

microtubules (pm) are seen along the entire pellic-

ular membrane with granular reticulum (gr) in

close proximity touching only microtubules which

are located directly under the flagellum. A

desmosome-like attachment (d) connects the flagel-

lum to the pellicle (P). X 63,000.

Figure 6. Trypanosoma lewisi, young form. Cross section

showing a distinct association between granular

reticulum (gr) and only sub-pellicular microtubules

(pm) located under the flagellar attachment zone

(far). ^5,000.

Figure 7« Trypanosoma lewisi. young form. Longitudinal

section through several flagella showing the

lattice-like arrangement of the intra-flagellar

structure, (if). X ^5,000.

2 5

pr

. i o x

1 . \

1 . \

l ! ' Is V - ' %

; 'i * "... ,

\ 'r-i / •%S

'"V_ ! c t .

« »

k * •"•

• - . 5

1 1 1

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• "' 'k Z "'f'^,-"-''-. Z*.. i * / • "' 'k Z "'f'^,-"-''-. Z*.. i r-, - v .

, — "-- * »>

'

V Vs- ^ ' .

. - - , -" .V

; f ^ ^ * -" ; 1 .

- / - V ^

r - , . "

i - C • 1

• ? " :"*' ^

' C S . .

W' 1 .

& - 4 W ' ^ H

M . . . " "-i, • ! ' V/Ac-> •-

'•'- ' J

^ _ - V

-- " ^ : s v

. ...

J * * 3 , ^ y i i " " -

s •• ' f t t \ « « I S ' !

„ y . • V. • '

k ^ J ™ - . . : - ? , k :,/ ^ ,-****

i

<*••* * * s- ^ ,

1 : H-4 F

: • # • ' 1

l a r j $

w > ^ J - £ 1

* " V : • = " .-.;5'' • * . . ' ' • * ' ' • ' V . ; *

y ^ y - ' > - » v > . . / > / U S* ' : . , - ' , . V;"v- >-. - . _ . .< V ?* ~ ' , V

^ - . f ^ . ^

i ' ; v . <- ' ' t „ " ,

1 I

, - ^ v - V '

• ? " ' / • • •

26

Figure 8. Trypanosoma iewisi, young form. Oblique section

demonstrating concentric arrangement of granular

reticulum (gr) with smooth-walled vesicles (sv)

secreted from the distal ends and more vesicles

of the same nature are free in the cytoplasm.

X 62,000.

O *7 ^ (

i ?*•>

Jr . %

V- $' ** gps# - , . . , ."Wft-.. '- • . w 1 ;>**-.*' j /-* - ,* -~**" /f""~ -"sT"'

- *'' - , "*< *V'* '• • "*; »- ' -"*** r ** cv -V-; ' "?'"i-V * - ? ' • " ' r >£?

1 'T '"-•:' " J * ' - **'

SfVjg •'• * - ;

rrf< :?•>....•% ^ -

» ? i ' * -_«;-• * "* i" v • „ , * " ?-'• '-»•• Si V-' 1

m m : & - * » ' • - • , 4 ' ; ;,: /.:- ••;** a^a:'*A:: - \ % • •' rv-.' . - i

.- *. ._ . ,• ^ . . - -• ^ Sj . . -c .; - 5 " 1* - J- _ . --v

fc?'% i < aV'i ^ ^ f.. > ,\ - - V;

rv m ^ , F : "';"-:4 *'

1 *' S¥ ' -*"-r" ' ;

• 1 i'"K' ;,. ./' * ' ' " :'" ' " ' "' " -*1' 'V'i' <-'. '='C ' /y-, • " " •- y % - . ••

-> '-}% ; :.i '

~1<J> -'r<

s ' *

^ - *

x'-- -

: " A-S^

^ "* rvv**-. . r> % f

> «. . "*-'

LJ .7 - *? : i v"- . •• - : •' ,- •*k\ - ".. ct-&$• * * -JV. t*." ^ r f : •-**--- '-^~hi*- >^-v±-y>- : .•"--•v,-. - -:•" - - , . - • -~z^\ ir- : •>< »•/ v ':- "" •- , " '5. r-r -v.--'-O -- J - ' ':;- - ' r-"%r '•>,«- •;. .-•••, v: • .-. "- ;r • •.. •- •- .:- -al . fejf'. -.• - -ii •-.••:• -•'& *:•

*>:•" %. -': *?>

• i-Xj.

A-A. -.i :-

8 S¥ "5..

' '"1- 1 ""I"-. ' "•;

| 'P>

-- -1>

28

Figure 9« Trypanosoma lewisi, adult form. Oblique section

showing granular reticulum (gr), tnitochondrium

(M), sub-pellicular microtubules (pm), flagellum

(F), irregular smooth-walled vesicles (iv).

Granular reticulum is attached to four sub-

pellicular microtubules located under the flagellar

attachment to the pellicle. X 62,000.

25

V . *-, *v . '- 'H' ' '• . . --

<•% ' *V-f j . *.*-i. s* > v^»._ -. t Vi - * ' % 5s

j> . -.

1 X ; - w

W l ; ^ !>' V > "t>;

I - - ; ^ a . -<- t r ' r .v

: j « r - - *

: }<S.

.» < * •«:•

: . % : . ? w & }

• '•• '' • ^ - v ^ v s W ' ' "

^ / . • ' " - v * s v * r *

' - %

- * - ; *,*,'*

# . si\ ^ - '/% "-. ' * - r * •&

X ?l

t r * i g

V =

r i v ^ \ V ', * " ; '4V- -t, ; - ; • - :'! Tv, c / ^ | K • • \

c \ V " <;•?.*>! 'ki, 5£J<

' '' r -jf^A-..

i k ."'

-

' v - : A- ^

; . n

'*^T '?r'

f ;"-

~<^' • \

: X

£ . v . i : ; H >-,;-•

- f t l ^ f,t- v --'.

* *-• "'"

! - • • . I V * , , J> ,• .

" . - ' V \ . :i % ---- ^

9 • / ••••', ' •-. " v '

•*•- - i *«•. -4

30

Figure 10. Trypanosoma lewisi, young form. Longitudinal

section showing five inclusion bodies (ib) lo-

cated in the posterior end of the organism in

close proximity to the flage Hum's emergence

from the cell. Other organelles seen in the

section are nucleus (N) with a dense and large

nucleolus (nu), nuclear membrane (rim), granular

reticulum (gr)» several dense and large gran-

ular s (g), vesicular Golgi complex (G),

mitochondrion (M), and smooth-walled vesicles

(sv). X 50,000.

s , • * *

^ s f f . / V , •rftm -

*r • '•** 'hr*

w , & .

' S f

f •. ' vl", - . --^'1 « _^s^" vS

' " **" V r> '" * *'%„

•:<i

W^MiM -v- , 1

>.

-,£K' - : " ^ '

*

, &"

H * -v ,% ^ *>* / ' - a •. -

: I

% , 1 * •J' V -.

;3;£<. ' i f '

"f

^ '

* /

\jr

fe. ~5S

I t

#Sf# r V ; :\ J?8*

. &"

«4

1 S!-'H-

\ • * - ' :L%i

/Mv-n * :- 4. v ! S b t ' S W ^ \£*.

32

Figure 11. Trypanosoma lewisi, adult form. Longitudinal

section showing a nucleus (N}» nucleolus (nu),

mitochondrium (M), agranular reticulum (ar)

terminating in sacs of secretion (sar) and sev-

eral dense granulars (g) varying in their

osmiophillic property are seen in the posterior

end of the organism. X 38,000.

33

? v-

-m #$%

KX&J-

r/Y4

?Kt'Wk£*. ,/t &£'•&%'• -*r?,y-' : • % - - ^ %' 'n imi % ^:f A i "' -•*•• >-,A. '

% ,~x - &

.« .. - Vvf,r: ' ' ^ - 7 ^ i# ""

%- \ '• %

y zt J? Y\$

£r ^ I . ,. i?: . ' . , { % - 1 ? * !

\A '"* *8gV - * '" V\ V

V * m's - y / /

A > *! j "•%»; *i> " *J V,

y*

Figure 12. Trypanosoma lewisi, young form. Gross section

through the anterior end of dividing organism

showing two nuclei (N) with nucleolus (nu) and

mitochondria (M). X 42,000.

35

3^- .£ssr

® 1 y - •* - ' " *'*

- v -*"' " ' ^

P " <' -fhi ' % 1 ' v * y- M

'-i. ? * • * z i ^ w -

s a g A ^ *4 v-W - > ^ ,*

. . . . , 3& &. ... •

v ^ £

- ^ •% •*. - M <* -

* * ' " " ' % # L 1 - 3?

2- -

- - J - r . -N l l t ' ^ t f Jf

X a 3 6 . , . ' " . - * • r

V " ' U

i ' f e

1 "-T.

I V / . : 4 * ' ; " * % -

>' ' W % * *

\ ^ - v %'* : j ... J 5 . " » „ ^ i ^ i s w Ji^SSEa&^SSaJa .

36

Figure 13• Trypanosoma lewisit adult form. Gross section

showing circular and bifurcated mitochondria

(M) and smooth membraned agranular reticulum

(ar). X 38,000.

3 7

r V i ;

. A 4 C 0>: £}%:

frv

- : ' v r 4 ' / J

J>:-Xy - ' v

£ • • - " * . • ' r - „ . •

*" - 4 x f " '-:4 '•&%

•%£-&rK-*y-:h - S - -.•?.• \ -• *'i i-

\

> : V - .

• : , " s | >

' > f

50.-^ f l * kM n

• ' B

f f | \ V - ' ^ C ' - i ! - - " I •

• ' ' • " • • " • - V ' - l i

: ' " ' s ;• ; •" : * . ; " - . 1

1 - V

,* j? •' •• V s . - : Mr -• :j >*'* - ' * • * * : < ' " : ' ' r •?. - ~ v r :

" V * y \ % \ ' 4 # : " V . - ' . . - ' i y •' • ? : 4 - V v

* • f - : 4 - |

l f > ! ' - m r ' - V

- \ \ ' % -f-l i;; / / J " ' - ' ." ••?.; ± r - t j -\ ?->• ' • ' -S - - J . i f / " V - - • • • . ' .'• - A . ' : •

••-%#*% - •/>.-

\ ; f &

r y . " <

? r 5

' V ^ ; s * ? • ;?

. .vr J

l l

\U

- '•«;•••' '"w

*•?

^ 4

m i ~k \ 1

's% jv

j . i

"

. 4 * /

; ;J.;V % ' ' t

- A s? , l !

. ' v - J

/ . J

, , i

38

Figure 14. Trypanosoma lewisi. young form. Oblique section

through the anterior end of the organism showing

granular reticulum (gr), mitochondrion (M),

nucleus (N), with large nucleolus (nu), and

nuclear pore (np). X 42,000.

3 9

e .

t o •' - < r - \ a

*\-',\ esv-^. _

i ^ ,

V' *1

W : .:'- r l l # 3 ^ ' - -',,• :•'-. * rl/;: "•'-.:'y: A -.i J | * j : i : " ' - v : ^ y % : . \ : g . , •^;-

* " W i %

^ A.. >

kO

Figure 15. Trypanosoma lewisi, young form. Oblique section

through the posterior end of the organism showing

the anterior portion of the kinetoplast (K), flag-

ellum (F), mitochondria (M), granular reticulum

(gr) smooth vesicles (sv), and inclusion bodies

(ib). X 30,000.

, J- ' V" ^ '

' / V - V - ' •

:"::U

- * ~ r -

\ r W -

. - f y : < 1

f . \ >

J 6

" = ; • • ' W $

%

f - k • >

H ;

-**-r >/ : : : & '

V > / -

' ; f " j V .

i M

- - o ' " '

' -

" ' : . -V

~:,-/

V ? , *

v

CCr'i r . / ;

_ 4 r -

- • ' & r : -

:

v-.

" -r '

% \ \

%> 4,\F

% y y — /

" O k ~ : 3 S . > . •

- v ,

K s k - s i f t n

1 4 ?

42

Figure 16, Trypanosoma lswisi, adult form. Oblique section

through the posterior end of the organism showing

a large mitochondrion (M) attached to the kinet-

oplast (K) by a mitochondrial canal. Several

dense granules (g) are seen in the same region.

X 17,000.

Figure 1?. Trypanosoma lewisi, adult form. Transverse sec-

tion through the middle of the organism showing

smooth tubule surrounding a vacuole. X 17,000.

Figure 18. Trypanosoma lewisi, adult form. Longitudinal

section through the posterior end of the organism

showing an elongated mitochondrion (M) extending

from the kinotoplast (K) to the anterior portion

of the cell. Numerous inclusion bodies are seen

in the posterior end. X 23,000.

•,>/" - >-• r ^''is£fm_.

_, *:WX&. - J?% ; " ' & S jS^x^ i% -

op;-, i-z.g.:'-- ..• - -• /-' •"' '" .-•^% ::;V" ;-** 4 ? "

r^n • w • *

1 ^ 1 ^v / -

5'.; • .;•• .. • •- r ,&*r J

ym>.

W'""' a

m^r-' \

m-m^< W-'

*5; r i. F" iWFl^ ^^ * #' r1*' ^ *-w,

" r / ^.=/ 4* % # *

;S 3

"?/ •%'

>lV t

^ %

&IC$

}'-"ri 8

f * . ? ^ vu,; I - u«-*>&&, - %. i 1 -5> "> M - .*' - .^: t<r'is

f. V \ ^^TeVi '-ft C% f.- ;: ->s

v" ®»-i'.-s"#f t g_ ¥ £V v s w» -iff - ^

i ^ y t i i y ^ : £*- IS? ^#-r '£.^-3

•."V ..-j, * * ".V '. , > -«>"~ *ij£~*~'/ <v

VU a. V- a"" > % • >

• ' v w-

'C\' "£&%: oJC ':$&*&!$•

/xi^T i '. -,v3

'* J V4 V-C;' r.t" . f

.-i'- --" "'' i^r

, -

- -f«' -'• -T

Jc0^ '

^ '

- "" ^ v

I-. 1 y *%"%S i*" : -i

- j m ^ , "*r - / ^

**

i&taaar-'--- r>*i ?r_ „ -«-j, -;ir,'*/'

'•<? '" "~£§t& - v5 '"I5/ \

t ' / : C r 3 • - ^r,

;

J -7 - % & *' $

Sti-itV-T ... . „

Ws>

Figure 19. Trypanosoma lewisi, young form. Oblique section

through the flagellar (F) origin and the kineto-

plast (K) (v/ith its dense fibers). Several

organelles are found in this region such as

mitochondria (M), granular reticulum (gr) and

several types of vesicles and granules scattered

throughout the cytoplasm (arrows). X 56,000.

Lb

- ; v - r * \ v

- <r fe

• , • • •

.A <&>

^-h% »tt-5

"V ; ; § P # ;

, ' V

;->r r •%

J N / * - ' v -' -t' -" '•<?% -J ''-n*ii" '

••-*? -w&mr 4 , *C ?- - '

T ; 4

- 0 : » f - - - . - v ' - . . ^ " - \ M / . V \ .

* - ' : • , v .

: : ? ; W * § V - V !

i ; " : " I ; ' ' I € m : : • < • * ' • • v ^ a

fe' - s*y ". • * n Ms-- . & ' - - 4 ^ ' : . $ v ^ \ • y - ¥ U i : - ^ '

W-:-^fr ^ * ' ,v *:•">& :-n\* i f f - ' V ' > • . ' ^ * * ? r ' • -*\ • * , - ; . 4

¥«• < - V -„ - - *••• " • -' a -15

-y • ; *- ^ k " K' v

r t r r - . - - v - : - v - ' W - ' . v

•A i<-'-

•k*- n , *; ' * '

J. 2 -' V " " •

y r ' - ; ' # 1 ;-•£ -

^ • ' ,

- W V \ 4 ™ 4 ; „ - J ' i / . ' ' / * r ' *'

% - * • : • ^ - ; ; . . ^ '

^ V.

K>i. .' — v

' -SrP -r ^ i1- ",".^

. U - :r

- V V/'^F * ' . J ' ^ ' --'>

, * v ' • - "•' ,- , j :

" ^ " " - v " - *.i r- -

s3z • ' T

f W W - . • s - 3 ^ •- •• •-*•' ' • I • > , f t f r ^ a '"

|r .. • r ' ..-

&

1 %- ; : •??>;: /

* 7 ; ^ 1 > V "

"S ' /'-? s. -r

JJ*$k

t 5 ' ' ' : ; " % ' V r r ^ .

* % m . . • -? -

y'"d£-^ -

I ^ - A

. - ' ^ v - ' ^ 1 , ; " . X / ; / ' - £

- - V ! ' \ -

:•• K ' •> o i "% - y \ " ' " - " v , .4

-., 4-$y^%-X- * : %,&-.• .

«i .4 . .. i-"? t - '. , \ . ; y A

...•• - . ,/ ^ ;,V."'.:-

'46

Figure 20. Trypanosoma lewisi, young form. Medial section

through the kinetoplast (K) and the flagellar

origin demonstrating detailed structures of both

organelles. The central flagellar tubulars (ct)

are seen extending beyond both kinetoplast plates

(KppKpg). Dense cytoplasmic matrix connect the

tubulars to the kinetoplast membrane. Several

other structures could be identified such as

flagellum pocket (fp), attachment zone (az) be-

tween the pellicle (P) and the flagellar membrane

(fm), and peripheral axoneme tubule (pt). The

kinetoplast (K) is divided into two distinct

parts, the anterior part which consists of a

tangle of dense fibers (kt) and the posterior

end which has a mitochondrial appearance with

cristae (cr). X 84,000.

li?

•fx _ , •X^

^ 't - .'j ~ '' v.:-- '

,f T:l:. * ' , ? ' : Y';,: J?

" f ar

six'

^ » " • fp

' ... * - •- -i: m& - ->•' .* "~*.J***?-Z'j- - *,- •

- ,,• %. . <", ^ . •- - P—*• .. :

*k j.: , • - - - ; •'•

V«

-*sf*y

:1

4: •* • - v~ -

. - v , s

' - .--Jit'" V - ;* * < - _*-a

fm

Pt kpi

kp2 ,*v f ~'~-W-i?<-. • Mr

• "" 'V K I T — g - — - ct *? S *

I ' ' #*• ' «

/ - > *3

lii «£%w\ .fi

•V 'I? ,} , ^

: / " " • *

v. - ?i'/ 4: p : -.

K

cn-*

ty-'cj . -*t.

•I-; ' / it '•-• >;•'' 1 ^ if#-: • • " -j f-ii y-v

•; !- ' '

^ y g -

i * :

-j. >

. . -

• T ' " -• \ • • • - - '

r

'j-'H - • i

DISCUSSION

In interpreting an electron micrograph there is always

the hazard of misrepresentation or distortion brought about

during the process of preparation of the organisms or tissues

for electron microscopy. During the course of this study,

micrographs obtained were constantly checked with results

from previous studies on other members of the genus Trypan-

osomatidae. The overall ultrastructure of both young and

adult forms of Trypanosoma lewisi shares several common

features with other trypanosomes, although some unique char-

acteristics have been observed.

The three-layered pellicle and sub-pellicular micro-

tubulars are characteristic features found in all other

species of trypanosomes. However, absence of a thick sur-

face coat on both young and adult forms of Trypanosoma lewisi

distinguishes them from other trypanosomes such as Trypano-

soma con^olense and Trypanosoma cruzi. Protective and

antigenic functions have been correlated with the presence

of a thick surface coat (50).

There is no evidence for the existence of a cytostome

in either form of Trypanosoma lewisi. Ultrastructure studies

on blood forms of Trypanosoma vivax, Trypanosoma congolense,

Trypanosoma equiperdum and Trypanosoma avium also indicated

4-9

the absence of cytostcme (1, 3» 50)» However, it has been

found in the posterior end of culture forms of '''gypanogoir;a

raiae, Trypanosoma mega, and Trypanosoma conorrhini (30, 35»

40}. The. presence of granular reticulum, mitochondria, dense

granulars, and vesicles in the vicinity of the flagellar

pocket suggests the involvement of this region in high meta-

bolic activity. With the absence of a cytostome in this

region I believe there is participation of the flagellar

pocket in cytostomal activity, as it has been suggested in

Trypanosoma congolense (50). Attachment of smooth-wall

vesicles to the flagellar pocket's pellicle is another evi-

dence supporting this hypothesis.

Specialized secretory cells in higher animals are char-

acterised by the existence of a conspicuous granular

reticulum system, where amino acids are assembled in long

chains of protein and elaborate Golgi apparatus, where pro-

tein is concentrated in compact vesicles after the addition

of carbohydrate moiety (6). Young forms of Trypanosoma lewisi

exhibit typical features found in higher animals' secretory

cells. Granular reticulum, vesicular Golgi apparatus, and

an abundance of smooth-walled vesicles are readily observed

throughout the cytoplasm of young trypanosomes. Change in

the Golgi apparatus from aggregates of vesicular complex in

young forms of Trypanosoma lewisi to a small lamellar complex

in adult forms, points out a sharp decrease in secretion

50

activity. The nature of the secreted materials is unknown,

although electron cytechemicai studios on Trypanosoma raiac

indicated the production of acid phosphate, a lysosomal

marker enzyme in the immediate vicinity of the Golgi appa-

ratus (35)«

The association of vesicles with the granular reticulum

in young forms of Trypanosoma lewisi exhibits a unique feature

which is not observed in adult trypanosomes. There is a

striking resemblance between granular reticulum found in

young trypanosomes and those found in Tetrahymena pyriformis

(10). Smooth-walled vesicles attached to distal ends of

Tetrahymena pyriformis' granular reticulum is associated with

fluid regulation by separating excess water from

Vickerma

agranular ret

tive and horro

An abundance

the cytoplasm,

n (50) connected the function of the complex

iculum attached to sacs of secretion with nutri-

onal production to be utilized inside the cell,

of such organelles in adult forms of Trypanosoma

lewisi suggests a shift in the nature of the secreted materials,

One of the new findings in this study is the penetration

of the central-tubulars of the axoneme deep into the cytoplas-

mic matrix. Previous studies on the flagellum structure of

several species of trypanosomes reported the termination of

the central tubulars in the kinetosomal zone. Presence of

the flagellum base in the middle of the flagellar pocket in-

creases the risk of damage caused by chemicals used during

51

fixation, dehydration, or embedding, In this study the period

of fixation was shortened to rc-cric3 the chance of damage

caused by osmium tetraoxide.

There is no evidence for the existence of filopodia in

either form of Trypanosoma lewisi. Recently, filopodia have

been observed in many species of trypanosomes (2*0. Several

functions have been associated with their presence, such as

antigen liberation, protection against antibodies, and attach-

ment (^9) •

D'Alesandro (9) linked differences between the two forms

of Trypanosoma lewisi in morphology, enzyme levels, rates of

protein and nucleic acid synthesis, and respiration, with the

indirect effect of ablastin, the reproduction inhibitory

antibody to trypanosoma. This study shows definite changes

in the overall ultrastructure, but I have not determined the

factor causing the transformation. The nature of inclusion

bodies and granulars found in the cytoplasm of both forms

of Trypanosoma lewisi cannot be resolved unless various elec-

tron cytochemical techniques are applied, such as those used

to localize acid phosphate in Trypanosoma gambiense (39)•

Finally, the results of this study coincide with data

obtained from comparative biochemical studies on young and

adult forms of Trypanosoma lewisii

1. Presence of large nucleoli in young trypanosomes

could be associated with high amounts of non-protein

nitrogen and RNA found in this form (19)•

52

2. Presence of a conspicuous granular reticulum and

an abundance cf vesiclcs in young trypanosomes

indicates high protein content in this form (19).

3. Glucose uptake and oxygen utilization have been

reported to be higher in adult trypanosomes (18).

This could be related directly to the presence of

more developed tubular mitochondria with numerous

cristae in these adult trypanosomes.

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