JHC4ESTIVE PROCESSES IN P HANARO.
207
Intra-cellular and General Digestive Processes
in Planarise.
By
O. Arnold,
From the Cytologieal Laboratory of the University of Liverpool.
With Plate 17.
IN 1878 Metschuikoff drew attention to the phenomena
of intra-cellular digestion occurring in Turbellarian worms.
Since that time but little has been published dealing with
this very interesting subject.
Metsehnikoff's short notice was followed by a paper by
Lankester dealing with intra-cellular digestion in the endoderm cells in the medusa oE Limnocodiurn, and two years
later, 1883, Metschnikoff published further observations on
intra-cellular digestion in the mesoderrn cells of Synapta and
Phyllirhoe.
Intra-cellular digestion has been observed in Ccelenterates
generally, sponges, Protozoa, and in the leucocytes of the
blood.
Withiu recent years several observers have dealt with the
digestion in the Protozoa, but apparently no work has been
published dealing with the cytological details of intra-cellular
digestion in any of the Enterocoela.
Mouton in 1902 and Nerinstein in 1905, following on the
earlier work of Greenwood and Saunders, have given long
and detailed accounts of the process of digestion in Amoeba,
Paramcecium, etc. These authors, however, have limited
their attention almost entirely to the intimate history and
staining reactions of the food vacuoles of those animals, and
their conclusions afford few data which shed any light on
208
U. ARNOLD.
the digestion in more highly organised animals such as
Planaria. Moreove:-, the methods of research are necessarily
different. In unicellular animals a considerable number of
facts may be ascertained by the observation of the effects of
intra-vitam staining. In animals such as the Planaria this
is impossible on account of their large size and opacity. The
observations here described have therefore been made upon
carefully preserved specimens, and the staining reactions are
therefore post-mortem.
The methods used were as follows:
A number of P l a n a r i a l a c t e a , which had been deprived
of all food of any sort for fifteen days (after which period of
time the cells of the intestine are entirely devoid of all food
remains, see fig. 11) were fed with fresh clotted pig's blood,
and fixed in Flemming's strong solution at various intervals
after feeding.
These intervals after feeding were as follows: j , ^, 1J, 3^,
27, 48, 52, 70, 76, 96, 118. When a Planarian has just fed,
the fixation is attended with difficulty owing to the fact that
immediately the animal is immersed in the fixing fluid it
contracts and ejects the recently ingested food with considerable violence, not through the pharynx, but anywhere through
the skin. If, however, the animal is cut into several pieces
at the same time that the fixative is poured upon it, this
difficulty is partially obviated, the whole procedure being too
rapid to permit of any violent contraction. Forty-eight hours
after feeding the lumen of the intestine is almost empty, most
of the blood having been ingested, and the Planaria fixed
after that interval did not eject any of the remaining contents.
The stains used were : (1) A triple stain—Basic fuchsin,
methylene blue and orange (r.,1 and (2) iron-alum-haamatoxylin, acid fuchsin and orange G-.
All the figures, except fig. 5, are drawn from preparations
stained by the former process.
1
I have given an account of the method of using this stain in a
paper on the "Ovi- and Spermatogenesis of Planaria lactea," ' Arch,
f. Zellforschung,' Bd. iii, Heft 3, 1909.
DIGESTIVE PROCESSES IN PLA.NAK.IiE.
209
Some cells fi'orn the intestine of a planarian which has been
without food for some seven or eight days are shown iu
PI. 17, fig. 13.
The cells of the intestine are of two sorts:
(1) Long, irregularly columnar cells.
The cytoplasm of
the cell (fig. 13) consists of a clear protoplasmic network,
enclosing several large vacuoles at its distal end, the vacuoles
towards the middle of the cell being smaller and fewer. The
proximal part of the cell consists of very much denser cytoplasm, in which the reticnlurn is very fine aud close, showing
an almost fibrillar structure at its extreme end.
The spaces
between the network take the acid stain, but the network
itself is stained by the basic colours, so that the proximal end
of the cell where the reticulnm is very dense is much darker
than the rest.
In this part lies the nucleus, which is small in proportion
to the cytoplasm.
The nucleus is round or ovoid, with a
deeply staining membrane, and a nucleolus which is staiued
bright blue by the methylene blue.
In an animal which has been starved for fifteen days the
vacuoles in the cytoplasm are more numerous and larger
(fig. 11). The cytoplasm around each vacuole is denser and
more granular than elsewhere, but a definite membrane
cannot be made out.
(2) Goblet-shaped gland-cells, only half as large, or less,
than the former, invariably with a small nucleus extremely
irregular iu outline, aud taking the basic fuchsin stain very
markedly. The cytoplasm is very granular, and is peculiar
in having a greater affinity for basic than acid stains,
staining as deeply as the nuclear material. I t is full of large
vacuoles, in which now and again is to be seen a residue also
stained by the tnethylene blue (figs. 11 and 13.)
ABSORPTION OF FAT.
W e will deal with the functions and history of these gland
cells first. There is generally one of them to every ten of the
210
tt.
ARNOLD.
others. When the intestine is empty they are large and the
vacuoles are full (fig. 11). Very soon after food has been
taken into the intestine the whole cell diminishes in size, till
at about the twenty-seven hour stage it is shrunken to a
fifth of its original size and quite flaccid (fig. 16). In this
condition it lies squeezed in between the columnar cells, so
much so that sometimes these cells appear to lie quite outside
the intestine, between the latter and the surrounding
parenchyma.
There can be little doubt that the gland cells secrete a
digestive ferment, which is probably used entirely for the
digestion of fat.
Within a quarter of an hour after feeding it will be seen
that the columnar cells are full of fat-globules, stained black
by the ostnic acid of the fixative (fig. 1). Even when the
lumen of the intestine is full of blood (red corpuscles, leucocytes, etc.) no fat-globules are to be seen lying free in the
lumen, nor Can any pseudopodial extensions oh the cytoplasm
containing fat-globules of the columnar cells be seen, suggesting that the fat has been ingested in an amoeboid fashion.
The gland-cells do not begin to return to their normal siae
till after about the forty-eighth hour, when almost all the
columnar cells are devoid of unaltered fat, and reach their usual
size again at about the seventieth hour. It is very noticeable
that no iugestton of solid particles ( i . e . true intra-cellular
digestion) takes place until the absorption of fat is over, and
the latter has undergone marked changes in the columnar
cells. A large part of the fat absorbed by the columnar cells
is digested in the cytoplasm of these cells, but some of it
is again passed out at their bases unaltered lying in the
parenchyma. The fate of these extruded globules will be
dealt with later on.
The fat first appears in the cytoplasm of the columnar cells
in very small globules, which by fusing together form much
larger ones, so that some cells within an hour after feeding
seem to be one mass of fat.
The researches of Munk, Moore and Rockwood, and others
DIGESTIVE PROCESSES IN PLAN ARIA!.
211
have shown that in the higher animals, especially io mammals,
the absorption of fat by the epithelial cells of the intestine is
brought about by the fat of the food being converted into
fatty acids and glyceriue by the action of lipolytic enzymes.
Only in that form can the fat be taken up by the epithelial
cells, which then again syuthesise the fatty acids into fat,
and the latter is seen in the cytoplasm of the cells in the form
of globules, being passed on by them to the lymphatic cells
and the lymphatic capillaries.
The process appears to be very similar in the Planariae,
and judging by the facts stated above, there is reason to
believe that the goblet-cells of the Planarite function as
organs secreting a lipolytic enzyme. Possibly they may
elaborate other secretions as well, but their ability to secrete
a fat-digesting fluid can hardly be doubted.
It has been pointed out that when the columnar cells are
full of fat-globules stained by the osmic acid, no such fat is
to be seen in the lumen. Ic was therefore necessary to see
whether there was any fat in the lumen in a form not acted
on by osmic acid. It is well known that the staining with
osmic acid is due to the presence of unsaturated compounds.
In view of the work of Lorrain Smith (307) it was thought
desirable to test the action of Nile-blue sulphate, which
staius not only the neutral fat, but differentiates the fatty
acids. For this purpose some Planaria were fixed a quarter
of an hour after feeding in a weak solution of foruiol and
cut with a freezing microtome. By this means all fat solvents,
such as xylol, etc., were avoided. The sections were then
stained for fifteen minutes in a strong aqueous solution of
the dye. In spite of the fact that the colour was slightly
masked by the blue colour taken by all the tissue, characteristic globules of fat in the columnar cells were seen, red to
reddish-yellow in colour. Care has to be taken not to
confuse loose red blood-corpuscles which have been shifted
from the lumen ou to the cells with these globules. The
colour is, however, entirely different, the fat-globules being
detinitely red under a high power lens, whereas the corpuscles
212
G. ARNOLD.
are yellow. Apart from this, fat-globules can be seen in the
cytoplasm of the endoderm cells, far too large to be mistaken
for any corpuscle lying over or under one of those cells. It
must be remembered that no ingestion of the corpuscles takes
place until some considerable time after the one hour stage,
at which these Planaria were killed.
This fact indicates, at least, that the fat which appears in
the eudoderm cells is a neutral fat, but whether the secretion
of the gland cells breaks down the fat of the blood into fatty
acids could not be ascertained, for the colour of the bloodcorpuscles completely masks any blueness which might be
present in the food magma.
However, sections of the one and a half hour stage, cut
in paraffin and stained with Nile-blue sulphate, showed a
definite bluish tinge in the magma, but not a trace of red.
The significance of this fact is important, for it shows that
the digestive process in Planaria is not, as has been stated by
Metschnikoff ('L'Immunite,' 1902), entirely intra-cellular, and
at the same time indicates the first step in tlie formation of
the highly complex digestive apparatus found in the higher
animals.
This first step is, we have seen, the production of a secretion by certain cells which enables fat to be absorbed. Such
cells are unicellular glands. If during the course of evolution these unicellulai- glands, instead of being diffused
thi-oughont the intestine, become aggregated in certain areas,
we are enabled to picture the formation of any of the multicellular glands which line the intestinal tract by the
subsequent invagination and enlargement.
Metschnikoff ('02) and in his work ' L'Immunite dans les
Maladies Infecteuses/ says that in Planaria digestion is
entirely intra-cellular, and this seems hitherto to have been
widelj' accepted.
Mesnil ('01) comes to the same conclusion in regard to the
Actinia, but this disagrees with the results of several other
workers.
Pratt's ('05) observations on the digestive organs of the
DIGESTIVE KROCUSSES IN l'LANARIJE.
213
Alcyonaria, lead her to conclude that large food bodies are
rapidly broken up into small particles, and in some cases
apparently acted on by some digestive ferment in the ccelenteron of the zooids before being ingested by the cells ot: the
ventral mesenterial filaments, and that " we have evidence
in the Alcyonariaa as in the Madreporaria of an intercellular
digestion by the secretion of a digestivefluidin the coelenteron
of the zooids, as well as an intra-cellular digestion which
occurs throughout the ccelenterates."
Jordan ('07) has come to similar conclusions on the digestion in Actinia, and says that in them digestion is both interand intra-cellular. He put little paper bags containing fibrin
in the gastric cavity of some Actinia, and found that the
contents were digested although the bags remained intact.
His results are in agreement with those of Krukenberg.
Even in Hydra, according to Hadzi ('06), an appreciable
amount of extra-cellular digestion takes place, the food being
slightly predigested in the lumen before being ingested by
the pseudopodia of the endoderm cells.
We need not be surprised then that in the more highly
organised Planarian, digestion is not entirely intra-cellular.
The alteration which the fat-globules undergo in the
columnar cells is characterised by very marked alterations in
their staining reaction. At first they are deep black owing
to the action of the osmic acid in the fixing fluid in which the
animals were preserved (figs. 1 and 2).
Each globule is enclosed in a vacuole. Within half an
hour after feeding, some of the globules at the free end of
the cell become paler, changing from black to grey, and then
brown. Within two hours after feeding (figs. 2, 4, and 5) the
change had proceeded a great deal further. The black
reaction to osmic acid is no longer present, and the fat takes
the less basic of the two basic stains, the fuchsin, till eventually it is only stained by the acid cytoplasmic stain, the
orange G. (figs. 4, 6, and 7). A vacuole is no longer visible,
and eventually the fat-globules are incorporated in the substance of the cytoplasm.
214
((. AKNOIiD.
INTKA-CKLLULAB DIGESTION.
After all the fat has been absorbed, and when all the
glHnd cells are empty (fig. 10), true intra-cellular digestion
(phagocytosis) commences.
The columnar cells push out at their free ends long pseudopodial extensions into the lumen of the intestine, and shortly
afterwards large vacuoles appear in which masses of red
blood-corpuscles are seen (fig. 3).
At this stage, one and a quarter hours after feeding, the
selective action of the columnar cells is very noticeable, for
only the red-corpuscles are ingested, but none of the leucocytes. The latter are ingested last of all, some forty-eight
hours after feeding (fig. 6).
The digestion of the red corpuscles takes place very slowly.
Kven ninety-six hours after feeding (fig. 8) they may be seen
intact in some vacuoles.
As digestion proceeds, the corpuscles lose their shape (fig.
6, b.c), till at last the vacuoles contain an amorphous mass of
particles, consisting chiefly of the envelopes of the corpuscles,
which are stained by the methylene blue.
The leucocytes are ingested singly, and a vacuolar space
soon appears round them.
The leucocytes which lie in the lumen of the intestine do
not appear to undergo any change at all. Even after fortyeight or fifty-two hours they can be seen scattered about iu
the lumen, their cytoplasm stained orange by the acid stain,
and the nuclear membrane and the chromatin in the nucleus
quite intact. But immediately a vacuole has formed round
them after ingestion (figs. 6 and 7, L.) their staining reaction
changes. The cytoplasm then takes a pink colour, due to
the basic fuchsin, their nuclear contents become diffused,
and shortly afterwards the separate chromatin masses are no
longer distinguishable (fig. 9).
This marked and rapid change in the staiuing reaction is
undoubtedly due to the fluid in the vacuole secreted by the
surrounding cytoplasm.
DIGESTIVE PBOCJBSSES IN PLANAKliE.
215
It is now a generally recognised fact that intra-cellular
digestion in Protozoa is accompanied by a secretion of acid
in the vacuoles. but with regard to the part played by this
acid in the process of digestion there is a large difference of
upinion.
Greenwood and Sauuders ('94) show that proteolysis commences when the acid reaction is over, and is replaced by a
neutral reaction.
That the vacnole fluid also contains a
proteolytic enzyme there can be no doubt.
Moutou ('02) succeeded in extracting from cultures of
Amoebae a diastase, chiefly of a proteolytic action and
approaching ti'3'psin in its nature. This diastase he identified
with the fluid in the interior of the digestive vacuoles.
Nirenstein ('05) does not think that the acid in the
vacuoles has anything to do with digestion, and Monton has
shown hy a most careful series of experiments that the
amoebo-diastase which he extracted from Amoeba? has a
digestive action in an alkaline, neutral or faintly ncid medium.
On the other hand, Metschnikoff ('L'linmunite'), by feeding
Plauaria with blood with which had been mixed some grains
of blue litmus, came to the conclusion that digestion in those
animals takes place in an acid medium.
" L'etude des planaires nous montre que la nourriture des
ces aniinaux subit exclusivement la digestion intra-celluhiire,
dans un milieu faiblement acide et avec l'aide d'un ferment
soluble. Elle nous fournit deja une preuve de ce que la
digestion intra-cellulaire typique est un processns chimique,
dfl a, ^'intervention d'enzymes."
I have shown in connection with the absorption of fat
that digestion in Planaria is not entirely intra-cellular, but
the sudden change in the staining reaction of the ingested
leucocytes is strong evidence in support of the view that the
intra-cellular digestion in these animals takes place in an
acid medium. The change in the staining reaction of the
cytoplasm of the iugested leucocytes from the normal acid to
the basic stains would seem to indicate that the ingested
material becomes impregnated by an acid fluid.
216
G. ARNOLD.
Occasionally, in even the earlier stages, some ingested
bacteria are seen, but they are not numerous. But in the
cells of two Planai-ia killed 118 hours after feeding they were
extremely numerous (fig. 10, b. and c ) , and also in the lumen
of the intestine.
At this stage the intestine is practically empty, except a
few masses of blood-corpuscles and leucocytes, with numerous
bacteria. That they appear in greater numbers only when
the food, or what is left of it, has been in the intestine for a
long period of time would suggest that the remainder of the
free food is undergoing putrefaction. No great importance
is to be attached to this isolated observation, but perhaps we
have here the indication of the formation of a definite
intestinal bacterial flora.
CHANGES IN THE NUCLEUS.
In all the columnar cells of the starved examples the
nucleus coutains only one nucleolus (figs. 11 and 13). At
the most active state of digestion (figs. 7 and 10) there are
two nucleoli, and sometimes even three. It is a question
whether this multiplication of the nucleoli is to be interpreted as an absorption of material from the cytoplasm to the
nucleus, or as an expression of increased activity of the
nucleus during digestion, with the consequent formation of
waste products.
EXCRETORY AND PIGMENT GRANULES.
In all the columnar cells of the intestine certain granular
masses are seen. They are highly refractive and preserve a
yellow colour independently of the staining (fig. 15). Most
of them are excretory products, but some can not be distinguished from the pigment granules which form the greater
part of the eyes of these animals. As digestion proceeds
they increase in number, but always occur in groups, and are
not evenly distributed through the cell.
DIGESTIVE PROCESSES IN PLANARIA.
217
PASSAGE OF FAT AND EXCRETORY GRANULES INTO THE
PARENCHYMA.
Some of the fat absorbed by the columnar cells is not
digested but passed out in globules at their bases into the
parenchyma (see fig. 2 ; on the i*ight a fat-globule is being
extruded).
These globules are taken up by some amoeboid
wandering cells (fig. 14), and also by the yolk-cells (fig. 12)
and the large parenchynaa cells (fig. 17). How these globules
reach the interior of the yolk-cells I have not been able to
ascertain. Any digestive power of an amoeboid nature in the
yolk-cells or even in the parenchyma-cells is extremely
unlikely. Nevertheless it is very striking that after feeding,
the yolk-cells which lie in proximity to the intestine are
crowded with fat-globules, whei'eas in unfed specimens the
yolk-cells contain scarcely anything but yolk-globules. After
feeding, fat-globules are numerous at the bases of the
columnar cells, and lying free in the meshwork of the parenchyma (fig. 14). The parenchyma-cells also contain numerous
excretory granules, massed together in vacuoles (fig. 17).
It would be expected that in an animal like Planaria devoid
of an anus, the excretory products would be shed into the
intestine to make their way out to the exterior by the
pharynx.
An examination of a very large amount of inaterial, consisting of some hundreds of slides, has afforded no evidence
in support of this view. Not only have I been unable to see
any extrusion of waste matter into the intestine, but a careful
search through numerous sections has failed to show any
trace of extruded excreta in the shape of the characteristic
yellow concretions in the lumen of the intestine.
Are they
so soluble that they are all removed when lying free in the
intestine by the pi-ocess of preparing the material for
sectioning ? If not, it is difficult to explain how they are
removed from the body of the Planarian to the exterior.
I wish to express my thanks to Dr. Roaf, of the Department
218
G. ARNOLD.
of Physiology in this University, for valuable advice on
staining for fat with Nile blue sulphate.
CONCLUSIONS.
Digestion in PI an a r i a lac tea, and probably in all
Triclads, is both inter- and intra-cellular.
The intercellular digestion is limited to fat. The fat is
broken down in the lumen of the intestine by the secretion
of the goblet-cells into fatty acids, which are then absorbed
by the columnar cells and synthesised again into neutral
fat.
Most of the fat is digested in the cytoplasm of the columnar
cells, but some of it is extruded into the parenchyma at their
base, and appears in the yolk-cells and in the wandering
cells.
The digestion in the vacuoles takes place in an acid medium,
as evidenced by the change in the staining reaction of
ingested leucocytes.
BIBLIOGRAPHY.
'78. Metsclmikoff, E.—" TJbev die Verdammgsorgane einiger snsswassertuvbellarieii," ' Zool. Anz.'
'81. Lankester, Bay.—" On the Iiitra-celluliir Digestion and Endodevm
Cells of L i m n o c o d i u m , " ' Quart. Jonm. Micr. Sci.,' vol. 21.
'83. Metschnikoffi, E.—" Untersuclmngen iiber die Infcracellulsire
Vevdauung bei Wirbellosen Tieren." ' Arb. Z. Inst., Wien,' Bd. 5.
'S6. Greenwood, M.—" On the Digestive Process in some Rluzopods,"
' Jonm. of Physiol.,' vol. vii.
'86. Krukenberg.—' Grnndziige einer Vei'gleichenden Physiologie der
Verdauimg.'
'89. Metsclmikoff, E.—" Recherches sur la, Digestion Intracelhilaire,"
'Ann. de l'lnst. Pastern-,' tome iii.
'01. Metsclmikoff, E.—' L'Inmiunite dans les Maladies Infectleuses.'
'01. Mesnil, M.—" Digestion chez les Actinies," ' Ann. de l'lnst. Pasteur.'
'02. Mouton, H.—]i Recberches sur la Digestion chez les Amibes,'
1
Ann. de l'lnst. Pasteur.'
DIGESTIVE PROCESSES IN PtANAlUiK.
219
'05. Nirenstein, E.—"Beitrage zur Erniihrungsphysiologie der Protisten," ' Zeits. Allg. Physiol.,' Bd. 5.
'05. Pratt, E. M.—" The Digestive Organs of the Alcyonaria and their
Relation to the Mesoglceal Cell-plexus," ' Quart. Journ. MicrSci.,' vol. 49.
'06. Hadzi, J.—" Vorversuche zur Biologie von Hydra," ' Arch. Entw.
Mechanik.,' Bd. 22.
'07. Jordan, H.—"Die Verdauung bei den Actinien," ' Arch. Gesamiute
Phys.,' Bd. 116.
'07. Lorrain Smith.—" On the Simultaneous Staining of Neutral Fat
and Fatty Acids by Oxazine Dyes," ' Journ. Phys. and Bact.,'
vol. xii.
EXPLANATION' OF PLATE 17,
Illustrating Mr. G. Arnold's paper on " Intra-cellnlar and
General Digestive Processes in Planarise."
All the figures, except 13, are drawn direct, using a 2 mm. oilimmersion Zeiss and 8 compens.-ocular. Figs. 9 and 15 with 18 compens.ocular. Fig. 13 « in. Swift and 6 ocular.
All the figures except 5, which is stained with iron-alum hssniatoxyliiiacid fnchsin ;uid orange G., are stained with the triple stain mentioned
in the paper.
FIG. 1.—A columnar cell from material fixed i hour after feeding.
F I G . 2.—
..
..
..
i „
FIG. 3.—
..
..
..
H hours
FIG. 4.—
..
.,
,.
3A ..
F I G . 5.—
..
..
..
H F I G . 6.—
..
..
.
48 .
F I G : 7.—
„
„
,.
52 „
FIG. 8.—Portion of a columnar cell fixed 96 hours after feeding,
showing pseudopodial ingestion of a leucocyte.
FIG. 9.—Portion of another cell, same stage as 8 (18 ocular).
FIG. 10.—A columnar cell from material fixed 118 hom-s after feeding.
FIG. 11.—A columnar cell and a gland-cell from an animal starved
for fifteen days.
220
G. ARNOLD.
Fia. 12.—A yolk-cell containing fat- and yolk-globules. Yolk
coloured blue.
FIG. 13.—Several columnar cells and one gland-cell from a Planarian
starved for five days. Normal appearance.
FIG. 14.—Meshwork of the parenchyma, showing a free fat-globule
and two anKeboid wandering cells, also containing fat. some of which is
undergoing alteration.
FIG. 15.—Excretory and pigment granules (18 compens.-ocular).
FIG. 16.—An empty gland-cell lying at the base of two columnar
cells, cf. fig. 11.
FIG. 17.—A parenchyma cell containing excretory granule^ massed
together in vacuoles.
/ . Osmicated fat.
af. Fat very much altered and partially
absorbed. I. Ingested leucocyte. he. Ingested blood-corpuscles.
bac. Bacteria, g. Goblet-shaped gland-cell.
Quart. Journ. After. Set. Vol 54, N.S. PI. 17.
Mr
'
8
INTRA-CELLULAR DIGESTION IN PLANARIA.