E. Ray (Edwin Ray) Lankester.

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Sometimes all the indi-
viduals, apparently quite
unaltered morphologically,




FIG. 13.

Agglomerated cluster of
male forms of TryjMnomor-
pha noctuae in the intestine
of the gnat. (After Schau-
dinn.)




Fro. 14.

A, binary union or agglomeration of T.
lewisi. B, primary rosette of the same parasite.
(After L. and M.)



become thus dispersed. At other times disagglomeration is only
partial, a certain number of the more feeble Trypanosomes remaining
together and slowly dying. If the agglomerating serum is very
powerful, however, or if the biological conditions remain unfavour-
able, the rosette does not break up and the parasites at length
die off.

The significance of the process has yet to be ascertained. By
some it is regarded as a purely involuntary proceeding on the part
of the parasites, and brought about more or less mechanically. A
suggestion put forward by Lignieres (54) is not without interest,
particularly when the recent work of Calkins on the essential
meaning of fertilisation is borne in mind. This author considers it
quite probable that, as a result of the close intimacy, a molecular
interchange goes on between the associated individuals, which may
have a stimulating or recuperative value.

C. Abnormal andlm ^.ution Forms. Involution and degenerative
phases of Trypanosonu have received attention and acquired an



220



THE HAEMOFLAGELLATES



importance altogether undeserved, owing chiefly to the fact that
many of these parasites have been studied, so far, only in strange
and unaccustomed hosts hosts to which they are unadapted, and
for which they, on their part, prove markedly pathogenic.

Trypanosomes appear to be, in most cases, able to support, for a
longer or shorter period, unfavourable conditions of environment,
whether due to the reaction of the host itself or to the transference
of the parasites to a strange medium. Sooner or later, however,
the organisms feel the effects of such changed circumstances and




Fia. 15.

Involution and degeneration forms of different Trypanosomes. A-E, T. gatribiensc (A, C, and
B after Bruce and Nabarro ; B and D after Castellani). F, K-P, T. brucii (F after Br. and
PI. ; K-P after L. and M.). G-J, Q and B, T. equinum (after Lignieres.) S, T. ftrucn, plas-
modial mass, from spleen pulp (after Br. and PI.).

become markedly altered. The strange forms and appearances
frequently described are probably for the most part l abnormal ; i.e.
they do not represent phases in the typical life-cycle, but are vary-
ing stages in a process of degeneration. Nevertheless, it by no
means follows that the parasites rapidly die off. On the contrary,
many of these involution-forms, on entering the blood of a fresh
host, are able to infect it, though they may even have been kept for
some time in artificial surroundings.

The course which involution takes varies in different cases, but
the process generally follows one or another of three lines, which

1 See footnote to p. 222.



THE HAEMOFLAGELLATES



221



may occasionally be met with in combination in any given abnormal
form, (a) Chromatolysis. Here there is either a more or less
complete loss by the nucleus (usually the trophonucleus) of its
chromatic constituents, which pass ,out into the cytoplasm leaving
only a faintly staining plastinoid basis (Fig. 15, A) ; or else direct
fragmentation of the nucleus occurs (F-j). 1 (b) Vacuolisation. The
frequent presence of a vacuole in many Trypanosomes, which is



A.




FIG. 16.

Involution and degeneration forms (continued). A-C, T. bnicii, after Br. and PI. ; D-G, T,
gamtnense, after Castellani ; H, T. brueii, after Martini ; J, K, T. equinum, after Voges ; L, T.
brucii, agglomeration-cluster, commencing to form a plasmodium, after Br. and PI.

probably to be regarded as a normal cell-orgaiiella, has been men-
tioned above. The first indication of abnormality in this direction is
perhaps afforded when the vacuole increases very greatly in size (Figs.

1 5, E ; 16, E). Other, irregular ones may appear in the cytoplasm,
when the involution becomes pronounced in character (Figs. 15, C;

16, G). (c) Change of form. This is, from the weird forms often
resulting, the most obvious line of involution. Alteration in shape is-
generally accompanied by an increasing loss of mobility. In the

1 In certain of these cases it is possible that something in the nature of chrom-
idial formation may be going on, leading to nuclear readjustment.



222 THE HAEMOFLAGELLATES

case of single forms the body becomes stumpy (Fig. 15, C-E), losing
almost entirely its trypaniform shape, and ends by being ovoid or
like a ball (c, H, L) ; l the flagellum is limp and inactive and partially
coiled up (j). In other cases, quite irregular multiplication occurs,
accompanied by incomplete cytoplasmic division, leading to the forma-
tion of distorted multinucleate and multiflagellate bodies (Fig. 1 6,
A-G). Lastly, various fusion -forms may be met with, masses of
Trypanosomes gradually losing their distinctness and constituting
large plasmodia (Figs. 16, L; 15, s), made up of a great number of
nuclei embedded in a common cytoplasmic matrix.

If the organisms remain subjected to the unfavourable influences,
or if involution has reached too advanced a stage, death and dis-
integration result. The cytoplasm is the first to disappear, becoming
hyaline and colourless, and refusing to stain up. The nucleus
rapidly follows suit. The most resistant elements are the kineto-
nucleus and flagellum, which may persist long after other traces of the
organism have vanished (Fig. 15, P), the former as a little thickening
at one extremity of the latter ; sometimes the flagellum alone is left.

5. MULTIPLICATION.

Binary longitudinal fission is, probably, of universal occurrence,
and appears to be the usual method of multiplication ; though
Trypanosoma leurisi, at any rate, possesses another method in
addition, namely, rosette -like segmentation.

The process of fission begins with the division of the nuclear
and locomotor apparatus, but the actual order of division of these
different organellae appears to be very inconstant and variable.
As a rule, the kinetonucleus leads the way, but sometimes the
trophonucleus may. The duplication of the flagellum begins at
its proximal end, that which is in relation with the kinetonucleus.
Until lately the process has always been considered as an actual
longitudinal splitting of the flagellum, following upon the separation
of the two daughter-kinetonuclei. Now and again examples are
met with in which the duplication of the flagella has taken place
before the kinetonucleus has divided. It seems probable that it
is really the division of the kinetonuclear centrosome which is the
essential prelude to the division of the locomotor apparatus. This
flagellar splitting has been described either as extending to the
distal end of the undulating membrane, after which the two halves
separate (Fig. 17, c), or as being practically limited to the root-

1 It is here, if anywhere, that there might be a possibility of regarding as involution-
forms phases which really belong to the normal life-cycle ; e.g. rounded-off, resting
phases (cf. the " resistant forms " of Holmes and others). In such, however, the
flagellum would doubtless be absent, while the nuclear elements and cytoplasm would
be as usual ; in fact, the parasites might well show a resemblance to the Leishman-
Donovan bodies (cf. pp. 255 et seq.).



THE HAEMOFLAGELLATES



223



portion, which becomes thickened and then divides, one half break-
ing away as a new, short flagellum, whose further growth is basal
and centrifugal (Fig. 20, D). Schaudinn found, however, that in




A. B

FIG. 17.
Stages in binary longitudinal fission of T. brucii. (After Lav. and Mesu.)

Trypanomorpha noctuae the whole of the flagellum, etc., is developed
quite independently from the daughter-kinetonucleus, and laid
down alongside and parallel with the old locomotor apparatus ;
moreover, Prowazek (I.e.) and also M'Neal (56) maintain that the
same is the case in T. lewisi. Nevertheless, in many cases it seems
hardly possible to doubt that there is actual splitting of the
flagellum ; where, for in-
stance, the two new flagella
of the proximal part of the
body appear to actually
join arid continue as one,
yet undivided flagellum
(as seen in Fig. 17, A
and B). Again, even
where a daughter-flagellum
is separate from the main
one, the course of the two
is often so exactly parallel
that their origin by longi-
tudinal fission is highly (After Prowazek.)

probable.

So far, we have not much knowledge with regard to the
cytological details of nuclear division. Prowazek has given a
description of the process in T. brucii. The kinetonucleus becomes
thickened and spindle -like (Fig. 18, A). Subsequently it becomes




Fio. 18.
Details in the nuclear division of T.



224



THE HAEMOFLAGELLATES



dumb -bell -shaped, after which the two halves become farther
separated, remaining connected only by a short thread (B).
The chromatin of the trophonucleus is arranged in eight rather
elongated chromosomes, which next begin to divide in a similar
dumb-bell-like manner (Fig. 18, c). The trophonuclear karyosome
(karyocentrosome) has frequently divided by this time; but in
one instance Prowazek observed it much drawn out and functioning
as an intranuclear division centre (D), the chromatin having
become aggregated around its ends.

In her account of T. raiae in Pontobdella Miss Robertson (I.e.)
has gone at length into the question of nuclear division. The
kinetonucleus appears to divide by a simple kind of mitosis though




Fia. 19.

A-D, stages in the binary longitudinal fission of T. eqainum ; K, multiple fission in the
parasite ; F and G, binary and multiple division in T. equiperdum. (After Lignieres.)



the details are extremely obscure. The trophonuclear division also
takes place by a simple kind of mitosis, but shows a well-defined
achromatic figure (comparable to a series of axial fibrils). This
probably arises from the trophonuclear centrosome. The figures
showing the later phases of the process convey quite the same idea
as does Fig. 12 of T. grayi. In fact, this case also appears to
conform to the same general plan as those above described.

The division of the cytoplasm takes place last. In the great
majority of forms this is equal or sub-equal, and the two resulting
daughter-Trypanosomes are of approximately the same size (Figs.
17 ; 19, c). Although the cytoplasmic fission usually begins at the
flagellar end, it may start at the opposite extremity (cf. Fig. 19, D).
In some instances (Fig. 19, E and G) the longitudinal fission is



THE HAEMOFLAGELLATES



225



multiple, the original individual giving rise, simultaneously, to three
or four descendants.

T. lewisi differs from most Trypanosomes in that the. cytoplasm,
generally * divides in a very unequal manner (Fig. 20). Indeed,
the process is more comparable to budding, since the larger or
parent individual may produce, successively, more than one




777.



F.



FIG. 20.



Unequal division in T. lewisi. w, parent-individual ; <1, daughter-individual ; (/*, daughter-
individual dividing, x 2000. (A-E after Lav. and Mesn. ; F after Wasielewsky and Senn.)

daughter-individual; moreover, the progeny may themselves sub-
divide before separating, the whole family remaining connected
together by the non-flagellate end (Fig. 20, E and F). In this type
of division, it may be noted, the kinetonucleus comes to lie alongside
the trophonucleus, or even passes to the other side of it (i.e. nearer
the flagellar end). This method of division forms, as it were, a

1 Swingle (81) has recently found that T. leu-lsi may also divide by equal binary
fission ; and in such cases the two flagella may lie on opposite sides of the body.

15



226



THE HAEMOFLAGELLATES



transition between binary fission and the other characteristic method
of T. lewisi, namely, segmentation or rosette-formation (Fig. 21).
The chief difference is that, in the latter, no parent-individual is
recognisable, the segmentation being equal and giving rise to a
rosette of equal daughter-Trypanosomes.

The small parasites resulting from either of these modes of
division (Fig. 21, E) differ from typical adults by their stumpy, pyri-
form shape, the position of the kinetonuclens near the flagellar end
of the body, and the absence, during the first part of their youth,
of an undulating membrane. At this period they have a somewhat
Herpetomonas-like aspect. These young individuals can them-




FIG. 21.



A-D, segmentation



(rosette- formation) in T. leitrisi ; in C nuclear division has finished and

the daughter-nuclei (of lx>th kinds) have taken up a superficial position, while the cytoplasm
has become lobulated at the periphery, prior to the formation of the daughter-Trypanosomes.
E, daughter-individual ; F, one dividing, x 1750. (After L. and M.)

selves multiply by equal binary fission, giving rise to little
fusiform parasites ; and, with growth, these gradually assume the
adult appearance.

6. THE LIFE-CYCLE OF TUYPANOSOMES

It may be safely said that this remains, even to-day, one of the
most difficult and most debated questions among the whole of the
Protozoa, in spite of the amount of work, of one kind or another,
which has been contributed to the subject during the last few
years. When the present writer compiled his Review of the
Haemonagellates (3) some years ago, Schaudinn's remarkable
observations had been, to all appearance, amply corroborated in
various directions by the testimony of the Sergents (77), Billet
(4, 5), Brumpt (9), Le"ger (50), and Rogers (94) ; in short, the



THE HAEMOFLAGELLATES 227

whole trend of research pointed at the time to a very complex
life-cycle of the Haemoflagellates and to a close connection with
the Haemosporidia. Since then, however, owing in a great^
measure to the work of Xovy and M'Neal on the Trypanosomes of
birds (62) and of mosquitoes (G3), the results obtained by
Schaudinn have become, to a large extent, discredited ; these
authors maintaining that they are capable of a quite different
interpretation. Moreover, influenced by their work on Insectan
Flagellates, Novy and his colleagues have gone to the other
extreme and expressed their belief not merely that Haemorlagellates
and Haemosporidia are entirely distinct, but also that the Trypano-
somes of Vertebrates do not undergo any true development or part
of their life-cycle in the Insectan host. This latter view, at all
events, is, we think, shown to be incorrect by the most recent
research, which, as above mentioned, seems all in favour of an
alternate, Invertebrate host, one of the most important indications
being with regard to the specificity of the latter a point of the
utmost consequence in its bearing upon investigations of this kind.
Leaving aside for the moment a consideration of Schaudinn's
celebrated memoir, it will be best to give first a brief account of
the results obtained in this connection by different prominent
researchers, to other aspects of whose work reference has been
previously made.

Dealing first with Trypanosomes of cold-blooded Vertebrates,
the earliest important observations are those of Leger (<">0), relating
to Ti'ypiuwpliisiiut Tcu'inni and Ti'>ip<.iiwsuina bitrl/aftilae of the loach.
L('-ger distinguishes ordinary ('indifferent") ami larger, more
granular (probably female) forms of the Ti'yptimyla&ma in the
blood of the fish. "When a leech (/A'y/mVryWx sp.) was allowed to
suck blood containing only these parasites, which thereupon passed
into its stomach, the indifferent forms degenerated and perished,
while the female ones became massive and showed nuclear changes,
preparatory, Lrger thinks, to a sexual process. At any rate, after
.some days, the intestine of the leech contained numerous little
narrow Trypanoplasms, of which some, very filiform, perhaps
represented male forms, while others possessed a kind of beak or
rostrum in place of the anterior flagelluin, which made them
resemble Trypanosomes. The development of Tri/panosoma bar-
batulae in another leech (Piscicoht) showed a certain amount of
agreement with that described by Schaudinn in the case of his
Avian Trypanosome in the gnat (Culex). Eighteen hours after
the leech had fed on blood containing exclusively T. barbatalae,
pyriform bodies lacking a flagellum (" ookinetes ") were found in
the intestinal contents. Some of these had a single large nucleus
(i.e. a compound nucleus) ; others had two nuclei, one smaller than



228 THE HAEMOFLAGELLATES

the other. Four days later the intestine contained numerous
Trypanosmes which could be readily distinguished as belonging to
one of Schaudinn's three types namely, indifferent, male, or female.
The male forms are very elongated and slender, provided with a
minute rostrum at the aflagellar end, and with a well -developed
flagellum at the opposite extremity, which renders them extremely
active ; they also creep or crawl with the rostrum in front. Their
cytoplasm is very clear and usually lacks granulations. Female
forms, on the contrary, are large and broad, with deeply staining,
usually granular cytoplasm ; the flagellum is only feebly developed
and the movement is sluggish. The indifferent individuals occupy
in most respects an intermediate position between the other two
types. A point of importance is that the kinetonucleus frequently
lies in about the middle of the body, and may be close to the
trophonucleus. There can be no doubt, it may be here remarked,
that these different sets of forms are of regular occurrence in, at
any rate, certain Trypanosomes. Since Schaudinn first described
them several observers have recognised them, in some instances in
the Vertebrate host, but always more sharply differentiated in the
Invertebrate. In general, the three types show the same charac-
teristics as noticed in the case of T. barbatulae. The indifferent
forms, Leger states, underwent active multiplication, by equal
fission ; those females which divided did so very unequally, by
a process somewhat like budding. The manner and form in which
the parasites passed back into the fish were not ascertained.

In his valuable contributions on the behaviour of Piscine
Trypanosomes in leeches, Brumpt (11) has noted developmental
phases of T. granulosum of the eel in Hemidepsis. Some hours
after arrival in the stomach of the leech, all the parasites become
pyriform, and by the position of the kinetonucleus close to the
trophonucleus recall L6ger's Crithidia-typQ (see below). By active
multiplication, an enormous number of little forms are produced,
which by the end of forty-eight hours have nearly all passed into
the intestine. Here they rapidly become elongated, assuming a
Herpetomonad-form, which may be retained for several months.
Some, however, by the end of seventy-two hours, have given rise to
true Tnjpanosoma-forms, with typical undulating membrane, which
pass forwards towards the stomach, and may be found accumulated
in the foremost stomach-coeca arid in the proboscis-sheath by the
fifth day. These are the forms which are inoculated into the eel,
becoming by simple elongation ordinary T. granulosum again.

Miss Robertson has published (72) some interesting observations
on a Trypanosome met with in Pontoldella muricata, which she
regards as T. raiae. This view is rendered extremely probable from
the fact that Brumpt (10) has found that T. raiae does develop in
Pontobdella. According to both authors the earliest phases occurring



THE HA EM O FLA GELLA TES 229

are rounded forms with both nuclei but no locomotor apparatus
comparable to ookinetes, in short (cf. T. barbatuhie above). These

individuals, says Miss Robertson, which divide in this condition

fairly actively, gradually disappear from the crop and are found
only in the intestine. Here they develop a locomotor apparatus,
but persist for some time in a Crithidia-likQ form ; they are of
varying size and may be very small. Later on, these individuals
take on a more or less typical Trypanosome-like, or, as we have
previously termed it, trypaniform character, with the kinetonucleus
in the aflagellar half of the body, though its actual position varies
greatly. These trypaniform individuals are of two main types,
which appear, however, to be connected by intermediate grades.
One kind is relatively very broad, with a relatively small kineto-
nucleus, but usually with a fairly long flagellum. The other type
is a long slender Trypanosome, with a large kinetonucleus, but the
free flagellum is not, as a rule, very long. The constitution of the
trophonucleus presents an unusual condition ; it is very much
drawn out, and the chromatin is arranged in a number of transverse
rods or bars (perhaps comparable to chromosomes) arranged more
or less parallel, like a ladder (cf. author's note on T. brucii above,
p. 216). About the middle of digestion, these Trypanosomes occur
chiefly in the intestine, but also in the crop, often in large numbers.
At a later period, a still more slender, practically thread-like form is
developed, which is met with chiefly in the proboscis, though also,
apparently, in the intestine. This type, which differs rather from
the last, appears to die off if it remains in the leech, and taking
this in conjunction with the occurrence of these individuals in the
proboscis, the inference is that this is the form in which the
parasites are inoculated into the fish. At the close of digestion,
a number of very small forms are always to be seen, either in a
rounded (probably resting) condition or in a very early Crithidial
phase. These seem to be persistent forms, through which the
leech retains the infection.

Miss Robertson discusses the likelihood of the two contrasting
trypaniform types above described representing male and female
individuals, but for several reasons hesitates to accept this view.
However this may be, it is more probable that conjugation itself
takes place soon after the transfer of the parasites from one host to
the other, i.e. after the arrival in the Invertebrate ; and that the
ookinete form is the immediate result of the process. This is
suggested by Leger's work on T. barbatulae, as well as by Keysse-
litz's account of the life-cycle of Trypanoplasma borreli (27). It
is also regarded by Prowazek (68) as being the case in T. lewisi, in
the louse.

According to Kcysselitz, male and female gametes can be
readily recognised in the blood of the fish (carp), the conjugation



2 3 o THE HAEMO FLAGELLATES

taking place in the leech, after various regulatory or matura-
tion processes have been undergone. The copulae give rise to
the three general types, distinguished principally by nuclear
differences.

In the case of T. lewisi, Prowazek states that soon after reaching
the mid-gut of the louse, the parasites undergo reduction of the
nuclear apparatus, by which the number of chromosomes is said
to be reduced from sixteen to four. The gametocytes (parent-
individuals of the gametes) are not strikingly differentiated from
one another, but in the formation of the microgamete from the
male form, the body becomes diminished in size, its nucleus
(trophonucleus) very elongated and at first spirally twisted, then
band-like, while also the cytoplasm stains differently from that of
the female element (megagamete).

Coming now to what is known of the development of Mam-
malian Trypanosomes in Tsetse -flies (Glossinae), we have first to
mention the knowledge obtained by Minchin, Gray, and Tulloch (59)
with regard to T. gambiense in G. palpalis. This, unfortunately, is
largely of a negative character, owing in all probability (as we have
seen earlier) to this species of fly not being the correct alternate
host, but one in which the attempts of the parasite to continue its
life-history are, for some reason, unsuccessful. Nevertheless, the
important observation that the types already recognised as male
and female in the blood of the Vertebrate at first greatly pre-
dominate, with, moreover, a much more marked differentiation
of sexual characters and without any forms intermediate in
type, is also strongly in favour of *,he idea that conjugation
occurs, in general, soon after the arrival of the Trypanosomes in
the insect.

No mention is made by Stuhlmann (80), in his highly interest-
ing account of T. brucii in G. fusca, of the occurrence of any similar
phases, or of anything in the nature of ookinetes, at the beginning
of the infection. The first individuals found by this investigator



Online LibraryE. Ray (Edwin Ray) LankesterA treatise on zoology → online text (page 24 of 32)