JHC4ESTIVE PROCESSES IN P HANARO. 207

Intra-cellular and General Digestive Processesin Planarise.

ByO. Arnold,

From the Cytologieal Laboratory of the University of Liverpool.

With Plate 17.

IN 1878 Metschuikoff drew attention to the phenomenaof intra-cellular digestion occurring in Turbellarian worms.Since that time but little has been published dealing withthis very interesting subject.

Metsehnikoff's short notice was followed by a paper byLankester dealing with intra-cellular digestion in the endo-derm cells in the medusa oE Limnocodiurn, and two yearslater, 1883, Metschnikoff published further observations onintra-cellular digestion in the mesoderrn cells of Synapta andPhyllirhoe.

Intra-cellular digestion has been observed in Ccelenteratesgenerally, sponges, Protozoa, and in the leucocytes of theblood.

Withiu recent years several observers have dealt with thedigestion in the Protozoa, but apparently no work has beenpublished dealing with the cytological details of intra-cellulardigestion in any of the Enterocoela.

Mouton in 1902 and Nerinstein in 1905, following on theearlier work of Greenwood and Saunders, have given longand detailed accounts of the process of digestion in Amoeba,Paramcecium, etc. These authors, however, have limitedtheir attention almost entirely to the intimate history andstaining reactions of the food vacuoles of those animals, andtheir conclusions afford few data which shed any light on

208 U. ARNOLD.

the digestion in more highly organised animals such asPlanaria. Moreove:-, the methods of research are necessarilydifferent. In unicellular animals a considerable number offacts may be ascertained by the observation of the effects ofintra-vitam staining. In animals such as the Planaria thisis impossible on account of their large size and opacity. Theobservations here described have therefore been made uponcarefully preserved specimens, and the staining reactions aretherefore post-mortem.

The methods used were as follows:A number of P l a n a r i a l ac t ea , which had been deprived

of all food of any sort for fifteen days (after which period oftime the cells of the intestine are entirely devoid of all foodremains, see fig. 11) were fed with fresh clotted pig's blood,and fixed in Flemming's strong solution at various intervalsafter 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 thatimmediately the animal is immersed in the fixing fluid itcontracts and ejects the recently ingested food with consider-able violence, not through the pharynx, but anywhere throughthe skin. If, however, the animal is cut into several piecesat the same time that the fixative is poured upon it, thisdifficulty is partially obviated, the whole procedure being toorapid to permit of any violent contraction. Forty-eight hoursafter feeding the lumen of the intestine is almost empty, mostof the blood having been ingested, and the Planaria fixedafter 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-haama-toxylin, acid fuchsin and orange G-.

All the figures, except fig. 5, are drawn from preparationsstained by the former process.

1 I have given an account of the method of using this stain in apaper 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 beenwithout food for some seven or eight days are shown iuPI. 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 vacuolestowards the middle of the cell being smaller and fewer. Theproximal part of the cell consists of very much denser cyto-plasm, in which the reticnlurn is very fine aud close, showingan almost fibrillar structure at its extreme end. The spacesbetween the network take the acid stain, but the networkitself is stained by the basic colours, so that the proximal endof the cell where the reticulnm is very dense is much darkerthan the rest.

In this part lies the nucleus, which is small in proportionto the cytoplasm. The nucleus is round or ovoid, with adeeply staining membrane, and a nucleolus which is staiuedbright blue by the methylene blue.

In an animal which has been starved for fifteen days thevacuoles in the cytoplasm are more numerous and larger(fig. 11). The cytoplasm around each vacuole is denser andmore granular than elsewhere, but a definite membranecannot be made out.

(2) Goblet-shaped gland-cells, only half as large, or less,than the former, invariably with a small nucleus extremelyirregular iu outline, aud taking the basic fuchsin stain verymarkedly. The cytoplasm is very granular, and is peculiarin having a greater affinity for basic than acid stains,staining as deeply as the nuclear material. I t is full of largevacuoles, in which now and again is to be seen a residue alsostained by the tnethylene blue (figs. 11 and 13.)

ABSORPTION OF FAT.

We will deal with the functions and history of these glandcells 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 thevacuoles are full (fig. 11). Very soon after food has beentaken into the intestine the whole cell diminishes in size, tillat about the twenty-seven hour stage it is shrunken to afifth of its original size and quite flaccid (fig. 16). In thiscondition it lies squeezed in between the columnar cells, somuch so that sometimes these cells appear to lie quite outsidethe intestine, between the latter and the surroundingparenchyma.

There can be little doubt that the gland cells secrete adigestive ferment, which is probably used entirely for thedigestion of fat.

Within a quarter of an hour after feeding it will be seenthat the columnar cells are full of fat-globules, stained blackby the ostnic acid of the fixative (fig. 1). Even when thelumen of the intestine is full of blood (red corpuscles, leuco-cytes, etc.) no fat-globules are to be seen lying free in thelumen, nor Can any pseudopodial extensions oh the cytoplasmcontaining fat-globules of the columnar cells be seen, suggest-ing that the fat has been ingested in an amoeboid fashion.

The gland-cells do not begin to return to their normal siaetill after about the forty-eighth hour, when almost all thecolumnar cells are devoid of unaltered fat, and reach their usualsize again at about the seventieth hour. It is very noticeablethat no iugestton of solid particles ( i .e . true intra-cellulardigestion) takes place until the absorption of fat is over, andthe latter has undergone marked changes in the columnarcells. A large part of the fat absorbed by the columnar cellsis digested in the cytoplasm of these cells, but some of itis again passed out at their bases unaltered lying in theparenchyma. The fate of these extruded globules will bedealt with later on.

The fat first appears in the cytoplasm of the columnar cellsin very small globules, which by fusing together form muchlarger ones, so that some cells within an hour after feedingseem 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 isbrought about by the fat of the food being converted intofatty acids and glyceriue by the action of lipolytic enzymes.Only in that form can the fat be taken up by the epithelialcells, which then again syuthesise the fatty acids into fat,and the latter is seen in the cytoplasm of the cells in the formof globules, being passed on by them to the lymphatic cellsand the lymphatic capillaries.

The process appears to be very similar in the Planariae,and judging by the facts stated above, there is reason tobelieve that the goblet-cells of the Planarite function asorgans secreting a lipolytic enzyme. Possibly they mayelaborate other secretions as well, but their ability to secretea fat-digesting fluid can hardly be doubted.

It has been pointed out that when the columnar cells arefull of fat-globules stained by the osmic acid, no such fat isto be seen in the lumen. Ic was therefore necessary to seewhether there was any fat in the lumen in a form not actedon by osmic acid. It is well known that the staining withosmic acid is due to the presence of unsaturated compounds.In view of the work of Lorrain Smith (307) it was thoughtdesirable to test the action of Nile-blue sulphate, whichstaius not only the neutral fat, but differentiates the fattyacids. For this purpose some Planaria were fixed a quarterof an hour after feeding in a weak solution of foruiol andcut with a freezing microtome. By this means all fat solvents,such as xylol, etc., were avoided. The sections were thenstained for fifteen minutes in a strong aqueous solution ofthe dye. In spite of the fact that the colour was slightlymasked by the blue colour taken by all the tissue, charac-teristic globules of fat in the columnar cells were seen, red toreddish-yellow in colour. Care has to be taken not toconfuse loose red blood-corpuscles which have been shiftedfrom the lumen ou to the cells with these globules. Thecolour is, however, entirely different, the fat-globules beingdetinitely red under a high power lens, whereas the corpuscles

212 G. ARNOLD.

are yellow. Apart from this, fat-globules can be seen in thecytoplasm of the endoderm cells, far too large to be mistakenfor any corpuscle lying over or under one of those cells. Itmust be remembered that no ingestion of the corpuscles takesplace 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 inthe eudoderm cells is a neutral fat, but whether the secretionof the gland cells breaks down the fat of the blood into fattyacids could not be ascertained, for the colour of the blood-corpuscles completely masks any blueness which might bepresent in the food magma.

However, sections of the one and a half hour stage, cutin paraffin and stained with Nile-blue sulphate, showed adefinite bluish tinge in the magma, but not a trace of red.

The significance of this fact is important, for it shows thatthe digestive process in Planaria is not, as has been stated byMetschnikoff ('L'Immunite,' 1902), entirely intra-cellular, andat the same time indicates the first step in tlie formation ofthe highly complex digestive apparatus found in the higheranimals.

This first step is, we have seen, the production of a secre-tion by certain cells which enables fat to be absorbed. Suchcells are unicellular glands. If during the course of evolu-tion these unicellulai- glands, instead of being diffusedthi-oughont the intestine, become aggregated in certain areas,we are enabled to picture the formation of any of the multi-cellular glands which line the intestinal tract by thesubsequent invagination and enlargement.

Metschnikoff ('02) and in his work ' L'Immunite dans lesMaladies Infecteuses/ says that in Planaria digestion isentirely intra-cellular, and this seems hitherto to have beenwidelj' accepted.

Mesnil ('01) comes to the same conclusion in regard to theActinia, but this disagrees with the results of several otherworkers.

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 arerapidly broken up into small particles, and in some casesapparently acted on by some digestive ferment in the ccelen-teron of the zooids before being ingested by the cells ot: theventral mesenterial filaments, and that " we have evidencein the Alcyonariaa as in the Madreporaria of an intercellulardigestion by the secretion of a digestive fluid in the coelenteronof the zooids, as well as an intra-cellular digestion whichoccurs throughout the ccelenterates."

Jordan ('07) has come to similar conclusions on the diges-tion in Actinia, and says that in them digestion is both inter-and intra-cellular. He put little paper bags containing fibrinin the gastric cavity of some Actinia, and found that thecontents were digested although the bags remained intact.His results are in agreement with those of Krukenberg.

Even in Hydra, according to Hadzi ('06), an appreciableamount of extra-cellular digestion takes place, the food beingslightly predigested in the lumen before being ingested bythe pseudopodia of the endoderm cells.

We need not be surprised then that in the more highlyorganised Planarian, digestion is not entirely intra-cellular.

The alteration which the fat-globules undergo in thecolumnar cells is characterised by very marked alterations intheir staining reaction. At first they are deep black owingto the action of the osmic acid in the fixing fluid in which theanimals were preserved (figs. 1 and 2).

Each globule is enclosed in a vacuole. Within half anhour after feeding, some of the globules at the free end ofthe cell become paler, changing from black to grey, and thenbrown. Within two hours after feeding (figs. 2, 4, and 5) thechange had proceeded a great deal further. The blackreaction to osmic acid is no longer present, and the fat takesthe less basic of the two basic stains, the fuchsin, till eventu-ally it is only stained by the acid cytoplasmic stain, theorange G. (figs. 4, 6, and 7). A vacuole is no longer visible,and eventually the fat-globules are incorporated in the sub-stance of the cytoplasm.

214 ((. AKNOIiD.

INTKA-CKLLULAB DIGESTION.

After all the fat has been absorbed, and when all theglHnd cells are empty (fig. 10), true intra-cellular digestion(phagocytosis) commences.

The columnar cells push out at their free ends long pseudo-podial extensions into the lumen of the intestine, and shortlyafterwards large vacuoles appear in which masses of redblood-corpuscles are seen (fig. 3).

At this stage, one and a quarter hours after feeding, theselective action of the columnar cells is very noticeable, foronly the red-corpuscles are ingested, but none of the leuco-cytes. The latter are ingested last of all, some forty-eighthours 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 seenintact in some vacuoles.

As digestion proceeds, the corpuscles lose their shape (fig.6, b.c), till at last the vacuoles contain an amorphous mass ofparticles, consisting chiefly of the envelopes of the corpuscles,which are stained by the methylene blue.

The leucocytes are ingested singly, and a vacuolar spacesoon appears round them.

The leucocytes which lie in the lumen of the intestine donot appear to undergo any change at all. Even after forty-eight or fifty-two hours they can be seen scattered about iuthe lumen, their cytoplasm stained orange by the acid stain,and the nuclear membrane and the chromatin in the nucleusquite intact. But immediately a vacuole has formed roundthem after ingestion (figs. 6 and 7, L.) their staining reactionchanges. The cytoplasm then takes a pink colour, due tothe basic fuchsin, their nuclear contents become diffused,and shortly afterwards the separate chromatin masses are nolonger distinguishable (fig. 9).

This marked and rapid change in the staiuing reaction isundoubtedly due to the fluid in the vacuole secreted by thesurrounding cytoplasm.

DIGESTIVE PBOCJBSSES IN PLANAKliE. 215

It is now a generally recognised fact that intra-cellulardigestion in Protozoa is accompanied by a secretion of acidin the vacuoles. but with regard to the part played by thisacid in the process of digestion there is a large difference ofupinion.

Greenwood and Sauuders ('94) show that proteolysis com-mences when the acid reaction is over, and is replaced by aneutral reaction. That the vacnole fluid also contains aproteolytic enzyme there can be no doubt.

Moutou ('02) succeeded in extracting from cultures ofAmoebae a diastase, chiefly of a proteolytic action andapproaching ti'3'psin in its nature. This diastase he identifiedwith the fluid in the interior of the digestive vacuoles.

Nirenstein ('05) does not think that the acid in thevacuoles has anything to do with digestion, and Monton hasshown hy a most careful series of experiments that theamoebo-diastase which he extracted from Amoeba? has adigestive action in an alkaline, neutral or faintly ncid medium.

On the other hand, Metschnikoff ( 'L'linmunite'), by feedingPlauaria with blood with which had been mixed some grainsof blue litmus, came to the conclusion that digestion in thoseanimals takes place in an acid medium.

" L'etude des planaires nous montre que la nourriture desces aniinaux subit exclusivement la digestion intra-celluhiire,dans un milieu faiblement acide et avec l'aide d'un fermentsoluble. Elle nous fournit deja une preuve de ce que ladigestion intra-cellulaire typique est un processns chimique,dfl a, ^'intervention d'enzymes."

I have shown in connection with the absorption of fatthat digestion in Planaria is not entirely intra-cellular, butthe sudden change in the staining reaction of the ingestedleucocytes is strong evidence in support of the view that theintra-cellular digestion in these animals takes place in anacid medium. The change in the staining reaction of thecytoplasm of the iugested leucocytes from the normal acid tothe basic stains would seem to indicate that the ingestedmaterial becomes impregnated by an acid fluid.

216 G. ARNOLD.

Occasionally, in even the earlier stages, some ingestedbacteria are seen, but they are not numerous. But in thecells of two Planai-ia killed 118 hours after feeding they wereextremely numerous (fig. 10, b. and c) , and also in the lumenof the intestine.

At this stage the intestine is practically empty, except afew masses of blood-corpuscles and leucocytes, with numerousbacteria. That they appear in greater numbers only whenthe food, or what is left of it, has been in the intestine for along period of time would suggest that the remainder of thefree food is undergoing putrefaction. No great importanceis to be attached to this isolated observation, but perhaps wehave here the indication of the formation of a definiteintestinal bacterial flora.

CHANGES IN THE NUCLEUS.

In all the columnar cells of the starved examples thenucleus coutains only one nucleolus (figs. 11 and 13). Atthe most active state of digestion (figs. 7 and 10) there aretwo nucleoli, and sometimes even three. It is a questionwhether this multiplication of the nucleoli is to be inter-preted as an absorption of material from the cytoplasm to thenucleus, or as an expression of increased activity of thenucleus during digestion, with the consequent formation ofwaste products.

EXCRETORY AND PIGMENT GRANULES.

In all the columnar cells of the intestine certain granularmasses are seen. They are highly refractive and preserve ayellow colour independently of the staining (fig. 15). Mostof them are excretory products, but some can not be distin-guished from the pigment granules which form the greaterpart of the eyes of these animals. As digestion proceedsthey increase in number, but always occur in groups, and arenot 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 notdigested but passed out in globules at their bases into theparenchyma (see fig. 2 ; on the i*ight a fat-globule is beingextruded). These globules are taken up by some amoeboidwandering cells (fig. 14), and also by the yolk-cells (fig. 12)and the large parenchynaa cells (fig. 17). How these globulesreach the interior of the yolk-cells I have not been able toascertain. Any digestive power of an amoeboid nature in theyolk-cells or even in the parenchyma-cells is extremelyunlikely. Nevertheless it is very striking that after feeding,the yolk-cells which lie in proximity to the intestine arecrowded with fat-globules, whei'eas in unfed specimens theyolk-cells contain scarcely anything but yolk-globules. Afterfeeding, fat-globules are numerous at the bases of thecolumnar cells, and lying free in the meshwork of the paren-chyma (fig. 14). The parenchyma-cells also contain numerousexcretory granules, massed together in vacuoles (fig. 17).

It would be expected that in an animal like Planaria devoidof an anus, the excretory products would be shed into theintestine to make their way out to the exterior by thepharynx.

An examination of a very large amount of inaterial, con-sisting of some hundreds of slides, has afforded no evidencein support of this view. Not only have I been unable to seeany extrusion of waste matter into the intestine, but a carefulsearch through numerous sections has failed to show anytrace of extruded excreta in the shape of the characteristicyellow concretions in the lumen of the intestine. Are theyso soluble that they are all removed when lying free in theintestine by the pi-ocess of preparing the material forsectioning ? If not, it is difficult to explain how they areremoved 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 onstaining for fat with Nile blue sulphate.

CONCLUSIONS.

Digestion in PI an a r i a lac tea, and probably in allTriclads, is both inter- and intra-cellular.

The intercellular digestion is limited to fat. The fat isbroken down in the lumen of the intestine by the secretionof the goblet-cells into fatty acids, which are then absorbedby the columnar cells and synthesised again into neutralfat.

Most of the fat is digested in the cytoplasm of the columnarcells, but some of it is extruded into the parenchyma at theirbase, and appears in the yolk-cells and in the wanderingcells.

The digestion in the vacuoles takes place in an acid medium,as evidenced by the change in the staining reaction ofingested leucocytes.

BIBLIOGRAPHY.

'78. Metsclmikoff, E.—" TJbev die Verdammgsorgane einiger snsswasser-tuvbellarieii," ' Zool. Anz.'

'81. Lankester, Bay.—" On the Iiitra-celluliir Digestion and EndodevmCells of L imnocod ium," ' Quart. Jonm. Micr. Sci.,' vol. 21.

'83. Metschnikoffi, E.—" Untersuclmngen iiber die InfcracellulsireVevdauung 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 derVerdauimg.'

'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 Pro-tisten," ' Zeits. Allg. Physiol.,' Bd. 5.

'05. Pratt, E. M.—" The Digestive Organs of the Alcyonaria and theirRelation to the Mesoglceal Cell-plexus," ' Quart. Journ. Micr-Sci.,' vol. 49.

'06. Hadzi, J.—" Vorversuche zur Biologie von Hydra," ' Arch. Entw.Mechanik.,' Bd. 22.

'07. Jordan, H.—"Die Verdauung bei den Actinien," ' Arch. GesamiutePhys.,' Bd. 116.

'07. Lorrain Smith.—" On the Simultaneous Staining of Neutral Fatand Fatty Acids by Oxazine Dyes," ' Journ. Phys. and Bact.,'vol. xii.

EXPLANATION' OF PLATE 17,

Illustrating Mr. G. Arnold's paper on " Intra-cellnlar andGeneral Digestive Processes in Planarise."

All the figures, except 13, are drawn direct, using a 2 mm. oil-immersion 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 hssniatoxyliii-acid fnchsin ;uid orange G., are stained with the triple stain mentionedin the paper.

FIG. 1.—A columnar cell from material fixed i hour after feeding.FIG. 2.— .. .. .. i „FIG. 3.— .. .. .. H hoursFIG. 4.— .. ., ,. 3A ..FIG. 5.— .. .. .. H -FIG. 6.— .. .. . 48 .F IG: 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. Yolkcoloured blue.

FIG. 13.—Several columnar cells and one gland-cell from a Planarianstarved for five days. Normal appearance.

FIG. 14.—Meshwork of the parenchyma, showing a free fat-globuleand two anKeboid wandering cells, also containing fat. some of which isundergoing alteration.

FIG. 15.—Excretory and pigment granules (18 compens.-ocular).

FIG. 16.—An empty gland-cell lying at the base of two columnarcells, cf. fig. 11.

FIG. 17.—A parenchyma cell containing excretory granule^ massedtogether in vacuoles.

/ . Osmicated fat. af. Fat very much altered and partiallyabsorbed. 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.