The Structure and Function of the AlimentaryCanal of Aplysia Punctata.1

By

• H. H. Howells, Ph.D.

(Department of Zoology, University of Bristol.)

With 14 Test-figures.

CONTENTS.

PAGE

I. INTRODUCTION 357

II . METHODS 358

HE. ANATOMY AND HISTOLOGY . . . . . . 359

Mouth and Buccal Mass, p. 360. Salivary Glands, p. 368.Oesophagus and Crop, p. 370. Gizzard and Filter Cham-ber, p. 372. Stomach and Caecum, p. 374. DigestiveDiverticula, p. 378. Intestine, p . 382. Rectum, p. 383.

IV. HYDKOGEN ION CONCENTRATION 384

V. DIGESTIVE ENZYMES . . . . . . . 384

VI. MODE OP FEEDING 387

VII. PHYSIOLOGY OF THE GUT 388

VIII. DISCUSSION 392IX. SUMMARY 395

I. INTBODUCTION.

THE species here described is Aplys ia p u n c t a t a Cuvier.The fullest accounts of the morphology and physiology of the

alimentary canal of Aplys ia are given by Zuccardi (1890),Mazzarelli (1893), Enriques (1901), and Eales (1921). The mono-graphs of Mazzarelli and Eales contain full reference to earlierliterature. With the exception of the section of the paper byEnriques devoted to the structure of the glandular epitheliumof the digestive diverticula, these accounts are all singularlylacking in detail and describe a uniformity of histologicai struc-

1 Owing to Dr. H. H. Howells's absence on active service, this paper,which represents the substance of the thesis he presented for the degree ofDoctor of Philosophy, has been prepared for publication by Prof. C. M.Yonge.

358 H. H. HOWELLS

ture which is not confirmed by critical examination. Moreover,the interpretation of the functions of the regions of the gut ismisleading. Eeference will be made later in this paper to otherwork which has been carried out on special problems relating todigestion in A p 1 y s i a.

The Opisthobranchs are very specialized in regard to theirfood and feeding mechanisms. Unlike P h i l i n e , S c a p h a n -der , H a m i n a e a , and Actaeon (Fretter, 1939), Aplys ia isstrictly herbivorous. An attempt has been made to correlate thefeeding mechanisms, mechanical action of the gut and the natureand distribution of the enzymes, with the food of the animal.

The research was conducted in the Department of Zoology ofthe University of Bristol and in the Laboratory of the MarineBiological Association at Plymouth. I am indebted to theUniversity of Bristol for the use of their table at Plymouth andto the Colston Research Fund of the University which defrayedcertain expenses. I also wish to thank Professor C. M. Yongewho suggested this research and offered much assistance andcriticism.

II. METHODS.The particular difficulty of obtaining suitable fixation of the

alimentary canal of Aplys ia has already been commentedupon by MacMunn (1899). The difficulty is made the more acutein the case of the glandular epithelium of the digestive diverti-cula owing to the extreme fragility of the cells which ruptureeven during the most careful excision.

Fixation of small pieces of the gut with Flemming (withacetic) preserved most of the cytoplasmic detail which could beseen in living cells, while the chrome-osmic mixture of Champygave almost equally valuable results. Adequate fixation oflarger pieces of the gut was obtained with Helly's modificationof Zenker's fluid and Ciaccio. Heidenhain's 'susa' and Bouinwere less satisfactory, differentiation of the cells being rendereddifficult by a partial solution of the cytoplasmic inclusions,while destruction of the glandular epithelium of the digestivegland was almost complete.

Sections from 4 to 10/x thick were stained with Heidenhain'siron-alum haematoxylin, followed by eosin, erythrosin or light

ALIMENTARY CANAL OP APLYSIA 359

green. For general purposes Heidenhain's 'azan' proved a mostuseful stain, offering consistent differentiation of the cytoplasmiegranules and clearly revealing the muscle fibres within thesub-epithelial connective tissue. Mayer's mucicarmine counter-stained with orange G- was used for the identification of mucousglands.

Fixation in neutral formalin followed by staining with pur-purin was employed for the detection of calcium deposits in thecells of the digestive diverticula. Fiemming •without acetic andCiaeeio's fluid were used to indicate the presence of fats andlipoids, Carnoy's fluid and absolute alcohol for that of glycogen.

The sites of absorption and secretion were determined withthe aid of soluble iron lactate introduced into the lumen of thegut and the blood stream respectively. Aplys ia could not beinduced to feed on the salt which was therefore forcibly pipettedinto the mouth. The presence of iron in the tissues was revealedby fixation in a mixture of equal parts of 5 per cent, ammoniumsulphide in 95 per cent, alcohol and Bouin, followed by treat-ment of the sections with solutions of potassium ferrocyanide(10 per cent.) and HC1 (1 per cent.). The sections were counter-stained in eosin or orange G.

III. ANATOMY AND HISTOLOGY.

According to Guiart (1901) the buccal mass, oesophagus, andcrop together comprise the foregut; the gizzard, stomach, andintestine the midgut; and the rectum the hindgut. He does not,however, state the evidence on which he bases this subdivision.The early development of Aplys ia has been described andfigured by Saunders and Poole (1910) who state that 'the•intestine grows out as a tube-like evagination from the rightposterior portion of the stomach. The anus is formed at once.and there is but a very slight ectodermal invagination—theoesophagus is long and narrow, and almost entirely endodermal.'Unfortunately they were unable to carry the larvae beyond thefree-swimming veliger stage. Later stages in the developmento f P h i l i n e a p e r t a are described by Brown (1924) but, apartfrom the buccal mass and salivary glands which are ectodermal,he gives no account of the embryonic origin of regions of the gut.

360 H. H. HOWELLS

It is therefore impossible to make any subdivision of thegut which has morphological significance, and for purposes ofdescription it is here divided into the following regions, distin-guished according to function: buccal mass, lodging the odonto-phore and into which opens a pair of salivary glands, oesophagusand crop; gizzard; filter chamber (corresponding to the ' secondtriturating stomach' of Zuccardi (1890), Mazzarelli (1893), andEnriques (1901), and the 'second portion of the gizzard' ofEales (1921)); stomach, bearing the openings to the digestivegland and caecum; and the coiled intestine bearing anteriorlythe opening to the stomach and leading posteriorly to therectum and anus which is situated on the posterior face of thesiphonal fold of the mantle. It must be emphasized that thisnomenclature does not imply either homology or identity offunction of these regions with those similarly named even inclosely related tectibranchs.

The gut is lined throughout by a simple columnar epithelium.This is ciliated in the oesophagus and crop, stomach, intestine,and rectum. In the buccal mass, gizzard, and filter chamber itbears a secretion which, tested by the modified Van Wisselingh-Brunswick tests of Campbell (1929), proves to be chitin.

Mouth and Buccal Mass.

The narrow slit-shaped mouth lies at the base of a shallowbuccal funnel (Text-fig. 1, bf) formed by the expanded bases ofthe anterior tentacles (%). The funnel is interrupted ventrallywhere it is separated from the foot by the shallow gutter of theopening of the pedal gland (jpg). The walls of the funnel carrya number of folds which extend inwards towards the mouth andare paler in colour than the general body surface. The epithe-lium of the folds is deeper (20 .̂) than that covering the generalbody surface and, except towards the base of the funnel,possesses an even dense carpet of cilia 7 \i long and arising fromdistinct basal granules. Yellowish brown pigment granules liewithin the cytoplasm towards the distal extremity of the cell.The epithelium is richly supplied with mucous glands of twotypes. Most numerous are the flask-shaped gland cells (Text-fig. 2, me), lying in a deep layer within the subepithelial con-

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362 H. H. HO WELLS

nective tissue, and discharging on to the surface by a coiled duct(dmc) running between the chitinogenous cells and terminatingin a small dilatation near the surface of the epithelium. Cells ofthis type occur in large numbers beneath the epithelium of thepedal sole. Here, in the buccal funnel, they undoubtedly lubri-

d-m.c.

m.c.

n.m.cTEXT-FIG. 2.

Part of a section through the dorsal wall of the buccal cavity, x 400.ct, connective tissue; ch, chitin; chc, chitinogenous cell; dmc, ductof mucous gland; me, mucous gland; mf, muscle fibres; nmc,nucleus of mucous gland.

cate the passage of food towards the mouth. The gland cells ofthe second type (Text-fig. 4, mc^} are situated in the epitheliumand are of the same height as the ciliated cells, with an expandeddeeper portion causing lateral constriction of the neighbouringcells and with a narrow distal extremity. They are, in thenomenclature of Hirsch (1931), polyphasic: the act of secretiondoes not end the life of the cell. The nuclei of both types {nmc)are large and rounded or irregular in shape, and contain aprominent nucleolus and chromatin staining heavily with iron-alum haematoxylin. The secretion is contained in vacuoleswithin a finely granular cytoplasm.

ALIMENTABY CANAL OF APLYSIA 863

• Towards the region of the mouth the ciliated cells give way tochitinogenous cells which are devoid of basal granules (Text-fig.2, chc). The funnel is here lined by a thin chitinous cuticlecontinuous with the substance of the 'jaws' (Text-fig. 1, j).The epithelium is here a little taller (23/x) and the intracellularnbrillae are strongly developed connecting from the base of thechitin, through the cytoplasm of the cell, to the basementmembrane. Beneath the jaws the fibrillae reach their maximumdevelopment and are distinguishable in the region of the ovalnucleus, about one-third of the length of the cell from the base.The upper portion of the cytoplasm bears a few refringentgranules, pale yellowish orange in life, and staining black withiron-alum haematoxylin after fixation in Ciaccio's fluid. Theyare probably of a lipoid nature. The jaws are local exaggerationsof the chitin supporting the rim of the mouth and strengthenedby a number of dark brown-coloured rods of harder chitin (the' stick cells' of Eales) packed closely side by side, and with theirfree tips slightly bent towards the buceal cavity. The rods areinterrupted at the dorsal and ventral corners of the mouth,giving the appearance of two distinct laterally disposed jaws.Behind the jaws the greater development of the cytoplasmicgranules obscures the intracellular fibrillae. Mucous cells of thefirst type are crowded in a wide layer beneath the basementmembrane and open into the buceal cavity through minutepores in the chitin.

The buceal mass lies immediately behind the mouth and infront of the nerve collar. At rest it is pear-shaped (Text-fig. 3);the narrower anterior region tapers towards the mouth opening,the larger posterior portion being more rounded. A low ridgeruns along the mid-dorsal surface from a point immediatelybehind the mouth to the oesophagus (o) which leaves the bulbon the posterio-dorsal side. The ridge overlies the dorsal foodchannel in the roof of the buceal cavity (Text-fig. 1, djc). Thetip of the radula sac (Text-fig. 3, rs) is visible externally as a lowrounded prominence, slightly less opaque than the adjacentmuscles, situated mid-ventrally. A shallow transverse groove(Text-fig. 3, Igr) separates the anterior and posterior regionslaterally, being interrupted dorsally by the median ridge and

364 H. H. HOWELLS

ventrally by a depression immediately anterior to the radula sacwhich receives the radular artery (Text-fig. 1 and 3 B, ra).

r.a.

l.gr.

TEXT-FIG. 3.

A, dorsal, B, ventral, and c, lateral, views of the buooal mass. In Bthe superficial intrinsic muscles I1 and P have been cut away toshow the underlying muscles. X 5. dsg, duct of salivary gland;E1 and E2, extrinsic muscles; I1-!6, intrinsic muscles; Igr, lateralgroove; o, oesophagus; sg, salivary gland (cut short). Otherlettering as before.

The pulley-like odontophore arises from the floor of theposterior cavity and bulges into the cavity filling it almostcompletely. Dorsally, the odontophore is divided longitudinally

ALIMENTABY CANAL OF APLYSIA 365

by a median groove which is shallow anteriorly but deepensrapidly into the radula sac. The radula (Text-fig. 1, r) is situatedon the sides of the groove, arising from paired depressions at thebase of the sac. New teeth are formed here. They are soft andcolourless and as they move forwards by the growth of theodontophore mature to a yellow and finally dark red colour.The worn teeth at the anterior edge of the radula are coveredby a tough white membrane (Text-fig. 1, m) which protects thewall of the buccal cavity when the odontophore is thrust for-wards to bring the functional teeth against the jaws.

The food channel runs the entire length of the roof of thebuccal cavity. When the buccal cavity is at rest the channel isenclosed by a pair of longitudinal folds (Text-fig. 1, dlf) formedby a greater development of the underlying connective tissueand closely approximated along their median edges. A numberof small colourless denticles project from the cuticle of the crestsof the folds bounding the anterior half of the channel. At theposterior end of the cavity the folds extend ventrally to meetthe ventral side of the oesophageal opening so that communica-tion with the oesophagus is only possible by way of the channel.

The buccal cavity is lined throughout by a thin chitinouscuticle, cilia being absent. The chitinous layer is finely striatedvertically with a broader horizontal banding which may repre-sent periodicity of secretion (Text-fig. 2, ch). In common withthe radula membrane and the newly formed soft teeth at thebase of the radula sac it stains pink with eosin and erythrosin,and a bright clear blue with ' azan'. It stains feebly or not at allwith haematoxylin. On the other hand the substance of the'stick cells', mature radula teeth, and denticles of the dorso-lateral folds stain intensely with haematoxylin and a brilliantred with 'azan'. The changes which ensue in the hardening ofchitin are therefore accompanied by well defined changes instaining properties. .Similarly, Sollas (1907) observed an in-creasing affinity for haematoxylin during the maturation of theradula teeth of H e l i x a s p e r s a .

The walls of the buccal cavity in contact with the sides of theodontophore consist of a spongy yellow tissue (Text-fig. 1, yt)containing gland cells the secretion of which lubricates the sides

366 H. H. HOWELLS

of the odontophore. The cells are of two kinds clearly distin-guishable in their staining reactions and cytoplasmic structure.

n. s. c. n.m.c.

BTEXT-FIG. 4.

A, section of a fold of the yellow tissue on the lateral walls of thebuceal mass, x 500. B, epithelial cells from the. same region.X 1500. mc-i, mucous gland; nclic, nucleus of chitinogenous cell;nsc, nucleus of secreting cell; sc, secreting cell; sgr, secretion gran-ule. Other lettering as before,.

Mucous cells of the intra-epithelial type already described(Text-fig. 4 B, mcij occur in large numbers. The deeper portions

ALIMENTABY CANAL OF APLYSIA 867

of the chitinogenous cells are typically much constricted toaccommodate the width of these cells which is sometimes as greatas 20/x. Outnumbering these are the secretory cells (sc), similarin shape to the mucous cells but with rounded nuclei andirregularly shaped secretion masses (the largest 5/A across)occurring singly or in clumps. The granules (sgr) stain red andblue with 'azan', the red coloration being limited to granules inthe neighbourhood of the nucleus (which lies near the basal endof the cell) and being progressively lost by the granules towardsthe distal end of the cell. The change in staining reactionpossibly accompanies the elaboration of the secretion mass.Fretter (1939) has described cells of an apparently similar kindin P h i l i n e , S c a p h a n d e r , and H a m i n a e a . From theirdistribution in these teetibranchs it would seem certain thatthey produce a lubricating secretion.

The musculature of the buccal mass (Text-fig. 3 A, B, C) is welldeveloped, the following intrinsic and extrinsic muscles respec-tively co-operating in its movements:

I1. A thin layer of muscle originating at the mouth region andradiating posteriorly over the surface of the anterior portion toits insertion in the lateral groove.

P. A thin superficial band encircling the posterior region. Itarises dorso-laterally from the groove and passes ventraily tocover the tip of the radula sac. Some of the fibres connect acrossthe mid-dorsal line roofing the buccal gutter. The band formsthe entire thickness of the wall dorsal to the attachment of theodontophore on ea'ch side.

P. Well-developed circular muscles lying beneath I1 and some20 times thicker than the latter. They form a sphincter aroundthe mouth and the anterior portion of the buccal mass. Themuscles are attached to ligaments on the mid-dorsal and mid-ventral lines.

/4. A thick mass of muscle originating in the groove anddiving beneath I2 to be inserted into the sides of the odonto-phore.

P. Paired fan-shaped muscles originating from the grooveventro-laterally and inserted into the sides of the radula sac.They lie beneath I2.

368 H. H. HOWBLLS

I6. A thick mass of muscle running predominantly in a trans-verse direction between the tissue constituting the so-called'cartilages' embedded in the halves of the radula pulley.

There are two pairs of extrinsic muscles (E1 and E2). Theyoriginate in the grooves and pass forwards and outwards to theirinsertion in the body wall around the mouth. Their fibres arecontinuous with those of the superficial intrinsic muscles P.

Sa l iva ry Glands .A pair of salivary glands (averaging 3 cm. in length, 2 mm.

in width, and 0-5 mm. in thickness) open on to the lateral wallsof the buccal mass below the folds of the buccal gutter (Text-fig. 1, sa) and discharge their secretion over the surface of theodontophore. The glands are attached posteriorly to theanterior end of the gizzard. A central channel some 0-14 mm.in diameter runs the entire length of each gland. It communi-cates by lateral openings with the acini, but some 3 mm. shortof its opening into the buccal mass it becomes a non-secretingciliated duct with a round lumen and a layer of circular musclessome 0-2 mm. thick (Text-fig. 3 A and c, dsg). A thin layer ofconnective tissue with scattered muscle fibres underlies theglandular epithelium of the acini. Secreting cells of two kindsoccur together with small groups of ciliated cells. The epithe-lium varies considerably in height and in the relative numbersof the three cell types in different parts of the gland. In theacini, where the epithelium is some 45/n deep, the gland cells aremost abundant. One type (Text-fig. 5, me), with a round heavilystaining nucleus and vacuolated finely granular cytoplasmstaining blue with 'azan' and a pale pink with muciearmine,probably secretes mucus which is poured out in masses. Theseextend for some distance between the cilia before finally break-ing free. The secreting cell of the second type (se) is typicallymore nearly cylindrical in shape and has a large nucleus con-taining a prominent nucleolus staining orange with 'azan'. Thevacuolated cytoplasm stains pink with 'azan' and is thicklypacked during life with pale yellow droplets, some highly re-fractile, and non-refractile granules. These are dissolved duringfixation. Ni (1933) observed the disappearance of these granules

AMMENTAEY CANAL OF APLYSIA 369

and droplets from the acini of the gland following secretion in-duced by electrical stimulation. The salivary secretion (at least

c.

cr. c

s.c,

n.m.c.

n.s.c.

TEXT-FIG. 5.

Portion of a transverse section through an acinus of the salivaryglands fixed in Ciaoeio, stained iron haematoxylin and erythrosin.The connective tissue has been drawn from sections stained'azan'. xlOOO. bg, basal granules of cilia; c, cilia; cic, ciliatedcell; mu, mass of secretion issuing from mucous gland. Otherlettering as before.

as obtained under experimental conditions) is heavily chargedwith droplets which, in view of the presence of a strong amylaseand a protease in extracts of the gland, are most probablyenzymatic in nature. The ciliated cells (cic) occur tightlywedged between the glandular cells. They bear long cilia arisingfrom small basal granules. The epithelium of the central duct

370 H. H. HOWBLLS

of the gland is somewhat shallower (36/x) and larger groups ofciliated cells occur between the secreting and mucous cells. Inthe short non-secreting portion of the duct the cells are muchshorter (12/u.) forming an almost cubical epithelium and bearinglong cilia

Oesophagus and Crop.The oesophagus and crop form a structural unit without

morphological differentiation. Viewed from the dorsal side theyform an S-shaped tube, the smaller, anterior loop bendingtowards the floor and the larger posterior loop towards the roofof the cavity. Two shallow annular constrictions situatedapproximately one-quarter and one-half its ; length from thebuccal mass divide the tube into three dilatations, each in-creasing in size posteriorly, and with wide connecting passages.The constrictions are never completely obliterated by the dila-tation of the crop, even when the animal is fully extended.Eales describes the presence of a constriction at the point ofjunction of the crop and gizzard. This is better described as asudden change in diameter between the thinner distensible wallsof the crop and the thicker, more rigid, walls of the gizzard, achange which is most marked after a meal.

The epithelium of the oesophagus and crop is longitudinallyfolded owing to variation in thickness of the underlying con-nective tissue. Some twelve folds are present at the narroweranterior end of the tube and are continuous in direction withfolds originating in the dorsal food channel. They increase innumber within the dilatations of the posterior crop and arecomplicated by a secondary longitudinal folding of the epithe-lium. These smaller folds alone can be seen when the gut is fullydistended, the primary folds being obliterated. The epitheliumclosely resembles that of the buccal cavity and dorsal foodchannel. The nuclei of the cells occur within the basal third ofthe cytoplasm the upper parts of which are packed during lifewith the yellowish orange refractile granules, accompanied bya few larger granules (Text-fig. 6, Ig) which are dissolved byCiaccio but precipitated by chrome-osmic fixation. A thin layerof cuticle (ch), identical in staining properties with the chitin of

ALIMENTABY CANAL OF APLYSIA 371

the buccal mass, lines the surface of the epithelium, but a finecarpet of threads arising from basal granules (bg) at the surface

i. cm.

n.m.c.

o.c.m.TEXT-FIG. 6.

Part of a transverse section through the wall of the crop, x 3000.bm, basement membrane; icm, inner circular muscles; Ig, granulesof a lipoid or fatty nature; Irn, longitudinal muscles; nmc, nucleusof mucous gland; ocm, outer circular muscles.

of the cell passes through the cuticle and apparently consists ofcilia (c). A cuticle in this position has been identified in theoesophagus of larval Act aeon by Mazzarelli (1906) and in theadult by Fretter (1939). It protects the surface of the epitheliumfrom coarse particles in the food. The cilia are extremely weak,being unable to move the smallest particles. Their activity mustbe quite subsidiary to that of the muscles in the functioning of

NOS. 331, 332 C c

372 H. H. HOWBLLS

the oesophagus and crop. Examination of living tissue provesthat they are not universally present in the epithelium. Mucouscells are very sparse, and in structure intermediate between thetwo types already distinguished in the epithelium of the buccalcavity. The enormous nucleus (Text-fig. 6, nmc), approaching20fi in diameter, lies towards the base of the flask-shaped cellwhich bulges the basement membrane (bm). Mucus is dis-charged through a narrow duct opening by way of a minute porein the cuticle. There is a layer of muscles (icm) attached to thebasement membrane, and this probably assists in folding whenthis portion of the gut is contracted. An outer layer of circularmuscle fibres (ocm), running in well defined bands, is separatedfrom the inner circular muscles by connective tissue throughwhich anastomose fine strands of longitudinal muscle fibres (Im).

Gizzard and F i l t e r Chamber .The outer circular muscles are best developed in the thick wall

of the gizzard where longitudinal muscles are absent. The innersurface of the gizzard is lined with chitin, and bears some 4 or 5alternating rows of teeth (about 50 to 60 in all) which representlocal thickenings of the chitin. Each tooth is pyramidal in shapeand secreted by a rhomboidal area of epithelium which is raisedabove the general level of the surrounding tissue. The anteriorfaces of each of the larger teeth have a greater area than theposterior faces, the apex of each tooth being posteriorly directed(Text-fig. 7). The anterior faces possess furrows (Text-fig. 7, ag)into which fit the edges of the pair of teeth lying on each side andin front. The teeth thus fit very closely and on contraction ofthe gizzard their points meet across the lumen except for thoseof the anterior two rows which are reduced in size. These fulfila particular role in the activity of the gizzard. The points of thelarger teeth (Text-fig. 7 A) are continually being worn down, theloss being made good by a secretion of new chitin at the base.The points of the teeth of the posterior row (Text-fig. 7 B) arenot worn down because they make no contact with the teeth ofother rows, and as a result they become greatly elongated andrecurved.

The musculature of the walls of the filter chamber closely

AMMBNTAET CANAL OF APLYSIA 373

resembles that of the crop. Here there are some 24 looselyarranged long acicular teeth (Text-figs. 9 and 10, d) each arisingfrom a small circular base.

The epithelium of the gizzard and filter chamber is composedentirely of chitinogenous cells identical with those lining the

«-§•

BTBXT-EIG. 7.

Triturating teeth of gizzard, A, tooth from the middle rows;B, tooth from the posterior row. x 12. ag, anterior grooves.

buccal cavity. An epithelium of a similar kind has been de-scribed for the gizzard of Cymbuliaperonii(Howells, 1936)and beneath the jaws ofHaminaea h y d a t i s (Fretter, 1939).The appearance of 'basal granules' at the surface of the cells,reported i n C y m b u l i a , i s here seen, with better fixation and_using critical illumination, to be due to the presence of minutepapillae of cytoplasm around each of the fibrillae as these leavethe cell to become embedded in the substance of the teeth. Theextra-cellular fibrillae hence form an anchorage for the secretion.But it is possible that their appearance may be due to a separa-tion of the epithelium from the secretion mass during fixation.They were visible after fixation by all the methods employed.Whether the intra-cellular fibrillae of the chitinogenous cell are

374 H. H.iHOWELLS

homologous with the fibrillae of the ciliated cell is also a questionwhich cannot easily be answered.

Stomach and Caecum.From its junction with the filter chamber the intestine

a. i.

- r e

ca.TEXT-FIG. 8.

Dorsal view of the alimentary canal posterior to the gizzard. Thedigestive diverticula have been dissected away to show thestomach and caecum, x c. 5. ai, anterior intestine; ca, caecum;fc, filter chamber; i, intestine; re, rectum; s, stomach bearing outends of the digestive diverticula.

describes a single loop within the posterior body cavity. Itsanterior portion (Text-fig. 8, ai) lies only partially embedded inthe digestive gland, the wall on the right side being visible atthe surface of the visceral mass. This portion, the 'intestinovalvolare' of Enriques, bears on its left side a large opening tothe stomach (Text-fig. 9, os) guarded by two folds of epithelium

AMMENTAEY CANAL OF AP1YSIA 875

(adf and vf) formed by the great development of the sub-epithelial connective tissue on the antero-dorsal and ventralsides. No special musculature is developed in connexion withthese folds. In their natural position (as seen through the trans-parent walls of the intestine) they completely obscure the open-

TEXT-FIG. 9.

Anterior intestine opened by a longitudinal incision along the rightside. The arrows indicate the direction of ciliary currents, x 6.adf, antero-dorsal intestinal fold; d, tooth of the filter chamber;/, faecal rod; os, intestinal opening of stomach; tx and 12, terminaldilatations of the major and minor typhlosoles; vf, ventralintestinal fold. Other lettering as before.

ing to the stomach. In dissection they do not retain their fullsize and the opening is revealed. It therefore appears that theshape and position of these folds is maintained by the pressureof blood in the sinuses they contain.

The so-called stomach (Text-figs. 8 and 10, s) representsanatomically a dilatation of the combined ducts of the digestivediverticula behind their opening into the intestine. It is com-pletely embedded in the tissue of the digestive diverticula.Large openings (Text-fig. 10, d'-d"") lead to the ducts of the

376 H. H. HOWBLLS

diverticula which divide almost immediately into narrowerducts, the number and position of which is subject to somevariation. The walls of the ducts are longitudinally folded, the

v.f.TEXT-FIG. 10.

The posterior filter chamber, anterior intestine, and stomach dis-played after being opened up by a longitudinal median incisionalong the right side of the filter chamber and anterior intestine,and a transverse incision across the floor of the stomach, passingthrough the aperture of the stomach into the intestine. Thearrows indicate the course of the ciliary currents on the walls ofthe stomach, d'-d"", ducts of the digestive diverticula; ec, ex-current channel of caecum; ie, incurrent channel of caecum;s, stomach. Other lettering as before.

folds converging on to the opening of the caecum on the posteriorsurface of the stomach (ic). From its junction with the stomachthe caecum (Text-fig. 8, ca) passes through the tissue of thediverticula, its distal end appearing on the right posterior surface

ALIMBNTAEY CANAL OF APLYSIA 877

of the visceral mass. Its cavity is incompletely divided longi-tudinally by two typhlosoles of unequal size (Text-figs. 10 and11, ti and t2) which extend beyond the opening of the caecumacross the posterior ventral wall of the stomach to terminate indilatations between the distal ends of the intestinal folds. Thefolds entering the caecum from the ducts converge on thechannel of the left side (here termed the incurrent channel(Text-figs. 10 and 11, ic) in view of its function) and are con-tinuous with the longitudinal foldings of its wall. The channelof the right side (excurr#nt channel) is smooth-walled. It isprolonged into a groove (Text-fig. 10, ee) separated from thecavity of the stomach by the extensions of the typhlosoles andit ends in the intestine. The typhlosoles terminate a shortdistance from the distal end of the caecum, thus affording acommunication between the right and left channels.

The epithelium of the stomach is similar to that of the ductsof the diverticula and the incurrent channel of the caecum. Itis a typical ciliated epithelium richly supplied with intra-epithelial mucous gland cells of the normal type and varying inheight (from 12 to 35/A) with the folding of the wall. Eibbons ofciliated epithelium continue beyond the ends of the ducts intothe glandular areas of the diverticula as previously observed inCymbul ia (Howells, 1936) and P h i l i n e (Fretter, 1939).

A different epithelium lines the excurrent channel of thecaecum (Text-fig. 11). The cells are taller (95ju.) and narrowerand consist of mingled gland cells (Text-fig. 12, gc) and ciliatedcells. The two occur in approximately equal numbers but thegland cells are much larger (see Text-fig. 12) and occupy thegreater part of the epithelium. The nuclei of the ciliated cells(ncc) are long and narrow while those of the gland cells (ngc) arerounded and occupy a lower level. In sections in which themucous gland cells of the incurrent channel are stained a brilliantred with mucicarmine the secretion of the gland cells of theexcurrent channel stains only faintly pink, while in sectionsstained with 'azan' the pale blue of these contrasts with theintense blue of the mucous gland cells. The secretion is there-fore not mucus but is probably a substance which imparts thefirm surface to the faecal rod. Droplets of the secretion occur

378 H. H. HOWELLS

abundantly between the cilia at the surface of the epithelium.Clear circular areas (iec) show up distinctly within the densercytoplasm beneath the nuclei of the gland cells. They, are toominute for their staining reactions to be ascertained with cer-

ec.

uc

TEXT-PIG. 11.

Transverse section of caecum, x 75. Lettering as before.

tainty. They are probably intra-epithelial canals which occurcommonly in the gut of molluscs where extra strength is requiredin the epithelium, e.g. in the style-sacs of Crep idu la (Mac-kintosh, 1925) and Os t rea (Yonge, 1926a), and in the caecumto the mid-gut of Cymbul ia (Howells, 1936). A thin layer ofmuscle fibres occurs in the connective tissue layer beneath theepithelium of the stomach, caecum, and ducts of the digestivediverticula.

Diges t ive D i v e r t i c u l a .The glandular epithelium of the digestive diverticula shows

marked changes of appearance according to the phase of activityof its cells. Pour types of cell may be distinguished. Most

ALIMENTARY CANAL OF APLYSIA 879

numerous are those rising to a maximum height of 75/4 above.the basement membrane, and with rounded ends projectingabove the general level of the epithelium (Text-fig. 13, abc). The

TEXT-FIG. 12.

Portion of a transverse section of the wall of the excurrent channelof the caecum. Fixed Hemming without acetic, stained 'azan'.X 500. gc, gland cell; iec, intra-epithelial canal; ngc, nucleus ofgland cell; yg, yellowish-green pigment granules. Other letteringas before.

cytoplasm contains numerous small round granules showing in-ternal granulation. The granules in the neighbourhood of the nu-cleus are coloured green in life (gg) and are slightly smaller thanthe brown granules (brg) which are crowded towards the free sur-face of the cell. The granules are coloured vivid green and redrespectively after treatment with 'azan'. The brilliant greencoloration is probably due to the action of the hot acetic acidin the azocarmine solution. MacMuna (1899) obtained a similarcoloration by heating in 33 per cent. HC1. The green granules

380 H. H. HOWBLLS

in the living cell were mistaken for chloroplasts by Enriques, butHorstadius (1933) has observed their formation from a diffusegreen pigment scattered throughout the cell immediately aftera meal and probably derived from the breakdown of chlorophyll.The cells have received the names of 'cellule absorbenti Chlora-

br.g.

s. c..ex. c.

TBXT-EIG. 13.

Part of a transverse section through a tubule of the digestive gland.Fixed Hemming without acetic, stained iron haematoxylin. Thecytoplasmic inclusions of the large cell are drawn from livingtissue, x 835. abc, absorptive cell; brg, brown granules; c4, cellof the fourth type; cr, orange-coloured crystal; ed, olive-greenexcretion droplet; exc, excretory cell; gg, green granule; «c4, nuc-leus of cell of the fourth type. Other lettering as before.

filliche' (Enriques), and ' Eesorptionszellen' (Biedermann andMoritz, 1899). But Horstadius has proved conclusively thatthese cells are incapable of phagocytic ingestion. Animals fedexperimentally with blood o fScy l l i umin the course of thisresearch failed to take up the erythrocytes while Ph i l inesimilarly fed showed an abundance of corpuscles within theingesting cells of the gland. Vesicles charged with browngranules may be found at various stages of separation from thesurface of these cells. The vesicles may break down later in thegut to liberate the granules which are found, together withunbroken vesicles, in the detritus bound in mucous strings on

AHMENTAEY CANAL OF APLYSIA 381

the wall of the stomach. Large orange-coloured crystals (er) ofhexagonal shape and unknown constitution occur occasionallyin these cells. That these cells absorb and excrete is thereforeapparent. Whether they perform a sceretory function in addi-tion has not been determined.

Secretion is the major activity of the cells of the second type(sc) which occur tightly wedged between the absorptive cells.The cytoplasm contams globular secretion masses staining blackwith iron-alum haematoxylin after fixation in Bouin. Enriquesreports the disappearance of these globules during digestion andtheir accumulation during starvation. Cells identical in appear-ance have been identified in Cymbul ia (Howells, 1986) wherethey were found to take up iron lactate from the blood-stream.

The cells of the third type (exc) are cone-shaped with a broadattachment to the basement membrane. They reach a maxi-mum height of approximately 4Q/x. The cytoplasm of thegrowing cell is charged with globules which collect to form a

' single large droplet (ec), olive-green in colour and surrounded bya thin layer of clear fluid within a vacuole some 25 fi in diameter,lying towards the distal extremity of the cell. The dropletconsists of a fluid of high surface tension which holds togetherwhen liberated into the lumen of the gut by the rupture of thecell. The cell is monophasic. The fact that the droplets arefound intact in the faecal rod within the caecum argues againstthe opinion expressed by Barfurth (1883) and by MacMunn(1899) that they are enzymatic in nature, and favours the viewthat they represent excrement.

The cells of the fourth type (c4) are very large and tightlypacked with colourless retractile spherules separated by thinlayers of cytoplasm containing minute yellowish brown gran-ules. The spherules are not dissolved by fixation with Ciaccio'sfluid, but do not resist chrome-osmic fixation. They stain a lightclear blue with 'azan'. In material fixed in neutral formalin aslight coloration with purpurin suggests that they contain alittle calcium salt, but fixation in this reagent is so unsatisfactorythat the preparations are almost valueless. Enriques reports(a) the disappearance of the spherules during fasting, the cellsbeing completely emptied after 10 days' starvation, (b) their

382 H. H. HOWELLS

solubility in water, and (c) the presence of similar spherules inthe cells of the diverticula of Cephalopoda. Enriques suggeststhat they are associated with the metabolism and storage ofcarbohydrate, presumably a pentosan, which has been identifiedin extracts of the digestive diverticula by Rohmann (1899) andBotazzi (1901).

All four types of cell occur throughout the glandular epithe-lium, the cells of the fourth type occurring in greatest numbertowards the distal extremities of the tubules. The tubules areclosely united by a thin layer of connective tissue containingscattered muscle fibres.

I n t e s t i n e .Posteriorly to the opening of the stomach the intestine (Text-

fig. 8, i) travels just beneath the surface of the visceral mass toreappear on the dorsal side. From this point it forms a singleloop around the anterior end of the mass, lying in a groove onits surface. Its walls are a transparent yellowish green in colourand provided with a thin layer of connective tissue and musclefibres, predominantly circular in arrangement. A flat strip ofepithelium occupying approximately one-fifth of the circum-ference runs the entire length of the tube. The remainder of thewall is longitudinally folded. The epithelium is ciliated andcolumnar. Over the intestinal valves it reaches an average depthof 80/x and bears a fringe of cilia 8/x long. The oval nuclei lie atvarious levels in the epithelium about the centre of the cell.Mucous cells (Text-fig. 14, me) of the normal type occur in theepithelium. In the intestine posterior to the valves the ciliatedcells are rather shorter (Text-fig. 14, cic), and a second type ofgland cell makes its appearance (sc) in approximately equalnumber to the mucous cells. The nuclei of these gland cells aresituated near the basement membrane, while the secretiongranules (sgr), staining brightly red with 'azan', lie within thecytoplasm towards the distal ends of the cells. When dischargedthe granules swell into thin-walled vesicles (sv) with a finegranular structure. They are carried away by the cilia andadded to the faecal mass, probably aiding the binding of theloose matrix of mucus and undigested food particles into the

ALIMENTARY CANAL OF APLYSIA 383

solid faeces. What may be designated basal cells (bad) occuroccasionally at the base of the epithelium. They have not beenobserved in contact with the surface of the epithelium. They

ci. c.

s.gr.

Kg.

c. m.

ba. c c-Im. s - c

TEXT-ITG. 14.

Part of a transverse section of the wall of the intestine. FixedBouin, stained 'azan'. x 500. bac, basal cell; cm, circularmuscle layer; sv, vesicle formed by the swelling of the extrudedgranules from the secreting cells. Other lettering as before.

are characterized by the possession of large nuclei each with aprominent nucleolus. Their function is obscure.

R e c t u m .The rectum (Text-fig. 8, r) is some 6 to 8 mm. long. It leaves

the visceral mass and extends dorsally within the connective

384 H. H. HOWELLS

tissue of the body wall to its opening at the anus on the posteriorface of the siphonal fold of the mantle. It is differentiated fromthe intestine by its smaller diameter, the absence of gland cellsother than mucous cells in its epithelium, and the greaterdevelopment of the circular muscles in its wall which form asphincter around the anus.

IV. HYDROGEN ION CONCENTRATION.

The pH of different regions of the digestive tract and itsassociated glands is given below, the results obtained agreeingclosely with those obtained by Yonge (1925). Fluid was ob-tained from the salivary glands after they had been washedrapidly in distilled water and teased out in a watch glass.Measured quantities of fluid were taken and added to indicatorson a white tile, the colours so obtained being compared withsimilar quantities of buffer solutions with indicators. The usualcorrections have been made for the salt error. The figuresrepresent the average results obtained from an examination ofsix recently fed individuals. A range of 0-4 was obtained in thefilter chamber, but elsewhere the figures agreed within 0-3.

pHBuccal mass . . . 6 - 2Salivary glands . . 6 - 0Oesophagus and crop . 4-8Gizzard . . . 4 - 9Filter chamber and anterior

intestine . . . 5-6

V. DIGESTIVE ENZYMES.

A number of investigations have already been made into thenature of the digestive enzymes ofAplysia . Inl899 Bohmannexamined the gut contents microscopically and noted the dis-solution of starch in the crop and the absence of this carbo-hydrate from the faeces. Botazzi (1901) obtained digestion ofstarch in v i t r o with crop fluid and extracts of the digestivediverticula. He also concluded, from the breaking up of thechloroplasts in the gut, that a protease is present capable of

StomachDigestive diverticulaCaecumPosterior intestine.Eectum

. 5-5

. 5-9

. 7-2

. 7-6

. 8-2

ALIMENTARY CANAL OF APLYSIA 38S

digesting the protein present in the chloroplasts. Enriques(1901) repeated the microscopical investigation in a number ofmolluscs and concluded that in Aplys ia the gut contains inaddition to an amylase and a protease, a cellulase which breaksdown the cellulose walls of the algal cells to liberate the chloro-plasts. Unfortunately, Enriques not only mistook the starchgrains for chloroplasts in the cells of U1 v a, but also the greenishbrown excretion masses from the diverticula for the chloroplastsin the gut fluid. The presence of a cellulase has not yet, as faras is known, been demonstrated experimentally in Aplys ia .The presence of an amygdaloclastic enzyme has been demon-strated by Giaja (1909) and of a saecharase by Bierry (quotedby "Vonk, 1937). Kriiger (1929) confirms the presence of anesterase in A. d e p i l a n s , and Horstadius (1933) a proteasecapable of digesting agar.

The nature of the secretions of the salivary glands and of thediverticula was tested experimentally and compared with thecomposition of the gut fluid. Extracts were made of the glandsby grinding the tissues (carefully separated from the adjacentviscera) with clean sand. The extracts were then made up to therequired strength with distilled water, bacterial action beingprevented by the addition of toluol.

The optimum pH of the activity of the enzymes was deter-mined at 30° C. The mixtures of extract and substrate werebuffered with the solutions of Mcllvaine and of Atkins andPantin. Eeducing sugars were quantitatively estimated by themethod of Hagedorn and Jensen (as modified by Boyland, 1928)and the products of protein digestion by the Sorensen formol-titration method. An estimate of the action of the lipase wasobtained by titration to pH 9 with N/100 NaOH using thymolblue as an indicator.

A strong amylase was found in extracts of the salivary glandsand of the digestive diverticula. The optimum pH for theactivity of the enzyme from the former source is at 5-6, and fromthe latter at 5-8, suggesting activity in the crop where a strongamylase is to be found with maximum activity at pH 5-6.Enzymes hydrolysing sucrose, lactose, and maltose are all activein the same region of the gut, being secreted by the digestive

386 H. H. HOWBLLS

diverticula. A glycogenase is active in extracts of the glands,but its absence from the lumen of the gut suggests that it is atissue enzyme. The conclusion is made the more likely by thenormal absence of this carbohydrate from the food.

Early experiments failed to demonstrate the presence of acellulase in the gut fluid or in extracts of the gut glands. Theaction of 10 per cent, extracts on finely teased Whatman filterpaper (no. 40) even after 14 days' incubation failed to producea detectable amount of reducing sugars. Using a high concen-tration of substrate, however, and a mixture of crop juice andbuffer solution in equal proportions, a weak action on filterpaper could be obtained after incubation for 30 days, its opti-mum pH being at 5-8.

Karrer and his co-workers, studying the digestion of celluloseby Hel ix found that for action on cellulose, cotton powder,cotton wool, filter paper, and various types of artificial silk, anundiluted or even concentrated digestive juice had to be em-ployed (for literature see Yonge, 1938). But when the crop juiceof Hel ix was submitted to the same experimental treatmentas that used for Aplys ia , an action many times stronger wasobserved. The results were confirmed by an examination of thecrop contents containing fragments of Ulva after incubationin v i t r o with a little toluol for 40 days. The cell walls wereintact, and the chloroplasts, although turned brown by theaction of the acid and collected together against the cell wallwithin the dead protoplasts, were still distinct. The polygonalcells were, however, more rounded in shape, small spaces havingappeared between them, possibly due to the weak action of thecellulase assisted by a more active digestion of an intercellularsubstance. In this connexion the identification of a pectinasein the diverticula and crop is of particular interest. The sourceof the cellulase is difficult to trace; no such enzyme could bedetected in extracts of the gut glands, possibly due to the factthat the enzymes were not sufficiently concentrated in theseextracts, although it is interesting to note in this connexionthat Yonge (1932) was unable to locate the source of the strongcellulase in the gut of P t e r o c e r a . The possibility of theexistence of a kinase from a separate source is suggested.

ALIMENTARY CANAL OF APLYSIA 387

The lipase in the lumen of the gut is no doubt secreted by thedigestive diverticula where an enzyme occurs with an optimumpH identical with that of the enzyme in the crop (pH 5-6), andcapable of digesting olive oil, rieinus oil, and esters. The esteraseaction is strongest, rieinus oil and especially olive oil beingdigested with greater difficulty.' These oils are present as largerglobules which present small surface area to the action of theenzyme.

A protease was identified in all regions of the gut, includingthe salivary glands, capable of attacking gelatin. Action onculcified milk was fairly rapid with extracts from the diverticulaand crop fluid, the enzymes present from these sources digestingfibrin, casein, and peptone. The protease was most activebetween pH 2-8 and 3-4 and also between 8-4 and 9-4. Sharpoptima were not found in experimental investigations of theprotease.

VI. MODE OF FEEDING.

Aplys ia was fed on a variety of weeds including species ofU l v a , Chy loc l ad i a , N i t o p h y l l u m , P o l y s i p h o n i a ,R h o d o m e l a , and Z o s t e r a . Starved animals will feed ontheir own egg cordons which they are, however, unable to digest.The small molluscs, annelids, and echinoderms occasionally tobe found in the contents of the crop have been swallowedtogether with the weed to which they were attached and do notform a normal constituent of the food.

The anterior portion of the foot assists in feeding. It firmlygrasps the weed, while the latter is explored by the walls of thebuccal funnel. The walls are freely movable, and when theymeet with a free end of the weed, they manoeuvre it into themouth. If no free end is encountered the weed is firmly grippedby the jaws, while the walls of the funnel proceed to shape theweed into a form capable of being passed through the mouth.

Contraction of the superficial intrinsic muscles I 1 and I2 andthe dorsal extrinsic muscles E1 pulls the posterior portion of thebuccal mass forwards. During this movement the organ losesits pear shape to become almost spherical and the posteriorextremity rotates forwards and upwards. A more pronouncedforward rotation of the odontophore occurs, operated by the

NOS. 331, 332 D d

388 H. H. HOWELLS

contraction of the basal mass of muscle I6 together with that ofthe muscles I4 attached to. the sides of the radula membrane.The latter cause a simultaneous divergence of the radular halves,the divergence being widest in front and spreading backwardstowards the radula sac at the completion of the motion. Thefunctional teeth of the radula are now firmly pressed against thejaws with their points raised owing to the increased curvatureof the membrane to which they are secured. After a slight pausethe contraction of the sphincter I 3 and of the ventral extrinsicmuscles B2 together with the relaxation of those muscles affect-ing the forward movement, returns the odontophore to itsformer position with a strong thrust. The movement is aidedby contraction of the muscles I5 attached to the radula sac. Atthe onset of the return movement the radular halves convergeabout the weed, but at the conclusion of the movement they lieapart in their position of rest. The weed is thus conveyedtowards the roof of the buccal cavity where it receives thesecretion from the salivary glands and is taken up by the lateralfolds of the dorsal food channel which firmly grasp the weedwith the aid of the denticles.

These movements draw in some 3 mm. of weed at each backward thrust of the odontophore. During these operations themouth is not completely closed. After some 2 cm. of weed havebeen taken in, a vigorous contraction of the sphincter during thereturn motion of the odontophore causes the jaws to tightenround the weed which is thus torn by the radula teeth. A p 1 y s i ais able in this way to feed comparatively rapidly. Large piecesof weed are taken into the crop.

VII. PHYSIOLOGY OF THE GUT.

Strong peristaltic waves of contraction originating at theanterior end of the food channel pass backwards to the neigh-bourhood of the second constriction, so propelling the weed intothe dilatations of the crop. Here it meets with digestive juicesregurgitated from the stomach, and with smaller pieces of weedwhich this fluid has flushed back into the crop from the gizzard.The posterior portion of the crop undergoes peristaltic contrac-tions timed apparently independently of those of the anterior

ALIMENTARY CANAL OF APLYSIA 389

region. These contractions force the weed against the teeth ofthe gizzard. The smaller teeth of the anterior rows grip the weedand ' stoke' it backwards into the mill formed by the succeedinglarger teeth. Their sharp tips diverge during dilatation of thegizzard and swing backwards as they converge on to the foodmass during contraction of the gizzard wall. They thus performan essentially similar function to that played by the threegroups of hard plates in the posterior crop of the larval Bul lah y d a t i s (Berrill, 1930). The weed is compressed between thelarger teeth as they approach one another and finally broken uptowards the end of contraction when the surfaces of the teethmove reciprocally by their slight rotation along a transverseaxis.

The muscular action of the walls of the stomach and of theducts of the digestive diverticula, acting together, drives thefluid secretion of the alveoli of the gland across the intestinalfolds (Text-fig. 9, adj and vf) into the anterior regions of the gut.Although the folds are very closely approximated, their surfacesare not in actual contact, so that fluid, together with the smalldroplets and granules of excrement which have escaped en-tanglement in the mucous strings on the walls of the ducts, passunhindered from the stomach forward as far as the crop. Theterm ' valve' for the intestinal folds is unsuitable, suggesting analternation of movements which have not been observed.Dilation of the stomach and ducts of the digestive diverticularesults in a passage of fluid from the crop through the gizzardand filter chamber. These pulsations are normally regular andcontinuous, and the observed movement of small air bubblesexperimentally introduced into the gut demonstrates theirvigour. The forward streaming of the digestive juices duringcontraction is strong enough to dislodge even large pieces ofweed from the gizzard and carry them forwards again into thecrop. On the other hand when expansion of the gizzard coincideswith expansion of the stomach, pieces of weed some 2-3 mm.across are frequently drawn through the gizzard and filterchamber together with food in a finely divided and fluid state.

The passage of material from the crop to the intestine istherefore slow. The presence of enzymes from the salivary

390 H. H. HOWELLS

glands and the digestive diverticula permit the digestion in thecrop of substances liberated from the plant cells by the ruptureof their walls in the gizzard.

A separation of particulate from fluid food is: effected byciliary currents operating in the anterior intestine. Text-figure 9illustrates the direction of these currents as followed by the aidof particles of weed taken from the crop and experimentallyintroduced into this region. Currents on the folds convey thelarger particles away from the opening to the stomach towardsthe bases of the folds. Here they come under the influence ofcurrents which convey pieces of weed directly from the filterchamber posteriorly into the intestine. The gut fluid, on theother hand, is drawn into the stomach through a ' filter' of ciliaat the approximated edges of the folds.

Cilia in the ducts of the digestive diverticula carry excretorygranules and droplets away from the alveoli into the stomach.

' Here cilia on the crests of the folds (Text-fig. 10) beat obliquelyinto the grooves in which this material is carried, in long mucousstrings, into the caecal opening on the left side. Here the thick,untwisted bunch of strings are conveyed by way of the incurrentchannel (Text-fig. 10, id) towards the distal extremity of thecaecum. They receive at the same time a generous coating ofmucus. At the end of the caecum the strings bend sharply roundand enter the excurrent channel. Here they are rotated in aclockwise direction and receive a layer of 'cement' from thegland cells (Text-fig. 12, gc) as they move back towards thestomach. It is possible also that the viscosity of the mucus isincreased owing to the lower hydrogen ion concentration in thischannel (Yonge, 1935).

A compact rod with a smooth surface thus issues from thecaecum and is conveyed between the typhlosoles across theposterior wall of the stomach into the intestine (see arrows inText-fig. 10). Here, under the influence of the cilia on the flatlongitudinal tract, it becomes spirally coiled, at the same timebecoming embedded in the looser packing of mucus and un-digested weed short-circuited from the filter chamber. Thecompleted faecal mass is finally consolidated and cemented bythe secretion of the glands in the intestine, while its passage

ALIMENTARY CANAL OF APLYSIA 391

through the tube is lubricated by a plentiful supply of mucus.The whole mass is propelled towards the rectum by the com-bined action of the cilia and muscles. The operation is probablyassisted by the higher viscosity of the mucus due to the lowhydrogen ion concentration in the posterior part of the intestineand rectum. A string of faecal pellets is ejected from the anusby the strong muscular activity of its walls, the constrictionsseparating the pellets being formed by contractions of the analsphincter. After a meal of Ulva the faeces are a strong greenin colour due to the presence of fragments of undigested weedwithin the matrix enclosing the spiral rod. Pieces of weed some3 mm. across, composed of cells unaltered by their passagethrough the gut, can easily be identified. The spiral rod, on theother hand, is a dark brown in colour and so compacted thatthe small yellowish green fluid droplets and even smaller pink,yellow, red, and green granules and greyish vesicles which canbe seen in the stomach are no longer distinguishable. In thefaeces of animals starved for 3 days it forms the only constituentand uncoils at the anus. It should be emphasized that the spiralrod is almost exclusively excretory and quite distinct in originfrom the undigested food residue in the surrounding matrix.

The study of the structure and function of the caecum inAplys ia has led to a reconsideration of views previouslyexpressed (Howells, 1936) as to the nature of the caecum in theThecosomatous Pteropods. This structure was originally notedby Souleyet (1852), while Meisenheimer (1905) showed that itwas universally present in this group and commented on itsresemblance to the style-sac in the Lamellibranchia. He furthersuggested that the rod-shaped mass it contains might have thesame function as the crystalline style. This was further empha-sized by Yonge (1926 b) who described the ciliary feedingmechanisms in a variety of these animals and showed that thefood is essentially similar to that of Lamellibranchs. Histo-logical examination of the caecum in Cymbul ia peroni i(Howells, 1936) revealed the great similarity between the epi-thelium of the caecum and that of the style-sac. But examina-tion of the caecum in Aplysia has revealed a similar structureand anatomical relations, while the function is clear. It is there-

392 H. H. HOWBLLS

fore concluded that the caecum in the closely allied Thecoso-matous Pteropods is not a style-sac but an organ concernedwith the consolidation and moulding of the faecal mass. It isinteresting to note in this connexion that in the P'otobranchiaYonge (1939) has recently shown that the ventral region of thestomach is similar in histological structure to the style-sac of theother Lamellibranchia and of certain Gastropoda, and it is alsosimilar to the caecum in these Opisthobranchia. This region inthe Protobranchia secretes mucus in which the indigestibleparticles are entangled and is responsible for the preliminarymoulding of the faecal mass. It does not secrete an amylase.It is suggested by Yonge that the style-sac with its containedstyle evolved from a caecum concerned with the secretion ofmucus for the consolidation of the faeces. Acceptance of thisview would have the additional advantage of explaining thesimilarity in histological structure between the caecum inA p 1 y s i a and in the Thecosomatous Pteropods and the style-sac.

VIII. DISCUSSION.Perhaps the most outstanding feature of the alimentary canal

of Aplysia is the.relatively enormous size of the anteriorregions in which food is mixed with the secretions of the diges-tive glands and digestion occurs. The so-called stomach ofAplysia is reduced, representing morphologically no morethan the combined ducts of the digestive diverticula immedi-ately behind their opening into the intestine. But the anteriorgut, comprising the oesophagus, crop, gizzard, and filter cham-ber, consists of a series of large dilatations possessing greatfreedom of movement within the anterior body cavity. A con-stant forward and backward motion of their contents is main-tained by the muscular action of the gut wall aided by theco-ordinated pulsations of the walls of the stomach and thedigestive diverticula behind. No distinction can be drawn,either morphological or functional, between the oesophagus andcrop when the animal is actively feeding. The entire length ofthe gut between the buccal mass and the gizzard is equallydistensible but, owing to the backward direction of the wavesof muscular movement of the gut wall, the anterior part of the

ALIMENTARY CANAL OP APLYSIA

tube is emptied more quickly. This then collapses to a narrowdiameter, thus simulating a purely conducting tube which hasbeen termed 'oesophagus' by earlier workers. The elaborationof the anterior gut is probably secondary. This is in markedcontrast to conditions in the primitive tectibranch Act a eonin which it is present as a straight narrow tube, without differen-tiation, between the buccal mass and the stomach, which isproduced into an extensive backwardly directed caecum on theleft side of the visceral mass. According to Fretter (1939), thestomach and caecum of A c t a e o n are probably of considerableimportance in digestion, the food here undergoing similar treat-ment to that received by the food in the anterior gut of morespecialized tectibranchs, i.e. trituration and admixture with thegastric fluid.

No successive action of enzymes from different sources of thealimentary canal is possible in A p 1 y s i a. All the enzymes acton the food in the same region of the gut. The fore-gut glandspour their secretion on to the food as it passes over the odonto-phore. The secretion contains, in addition to the mucus whichis a normal product of these glands in molluscs, a weak proteaseand a strong amylase. The food and salivary secretion isimmediately conveyed into the crop where it meets with a strongamylase, sucrase, lactase, maltase, pectinase, and proteases,together with a weak cellulase, secreted by the digestive diverti-cula and regurgitated by the rhythmic contractions of thetubules of the gland and of the walls of the stomach.

It is surprising that an exclusively herbivorous mollusc shouldpossess a cellulase which, acting alone, is not strong enough toliberate the contents of the cells of the weed upon which theanimal feeds. The action is many times weaker than thatobserved for the crop contents of Hel ix under the sameexperimental conditions. The triturating action of the gizzardof Aplys ia is, however, very efficient and is almost entirelyresponsible for the exposure of the contents of the plant cells tothe action of the enzymes. The food receives only very slighttrituration in the buccal mass. The weed is torn into pieces ofcomparatively large size (often 1-5 cm. long) when a contractionof the oral sphincter coincides with a backward thrust of the

394 H. H. HOWELLS

odontophore. The chief action of the radula is that of a con-veyor belt, rapidly passing food from the mouth to the roof ofthe buccal cavity where it is immediately seized by the sharpdenticles on the longitudinal ridges bounding the muscularbuccal gutter. The importance of a rapid feeding mechanism toan animal which has to deal with a comparatively large quantityof food and which is confined to an environment in which feedingis often made impossible by the strong action of tidal currentsis evident.

In marked contrast with that of Ae taeon , the caecum ofA p 1 y s i a is concerned solely with the cementing into a compactfaecal mass of the material excreted from cells in the digestivediverticula. A similar structure occurs in closely allied genera,e.g. A c 1 e s i a, and in the Thecosomatous Pteropods, in all ofwhich there is a similar need for avoiding the fouling of ciliarymechanisms lying in the path of the faeces after they are voidedfrom the anus. In other tectibranehs (e.g. P h i l i n e , Sca-p h a n d e r , G a s t r o p t e r o n , and Aetaeon) a spiral pallialcaecum (the 'caecum glandulaire' of Pelseneer (1894) and the'hypobranchial gland' of Brown (1934)), lies near the anus. Itis lined by mucus-secreting cells and strips of strongly ciliatedepithelium which draw a vigorous current of water over the anusand into the caecum. The current is thence expelled through theexhalant aperture at the posterior end of the mantle cavity. Inthis way material voided by the anus is rapidly conveyed awayfrom the mantle cavity. Nothing corresponding with this pallialcaecum is present in the mantle cavity of Aplys ia and theThecosomatous Pteropods. The respiratory current in Aplys iais created by the cilia on the etenidium and is sluggish in com-parison with that of the tectibranehs cited above. The faecalpellets remain in the region of the anus for a considerable timebefore eventually falling clear of the mantle cavity. In theThecosomatous Pteropods the faeces are voided into the reducedmantle cavity on the right side of the visceral mass. They thenpass forwards and leave the body in the immediate neighbour-hood of the ciliary feeding tracts.

• ALIMENTARY CANAL OF APLYSIA 395

IX. SUMMARY.

1. The anatomy and histology of the alimentary canal, pro-cess of feeding, and physiology of digestion in Aplysiap u n c t a t a have been investigated.

2. The food undergoes little trituration in the bueeal cavity.The mode of action of the jaws and odontophore is adapted tothe rapid intake of vegetable food.

3. The oesophagus and crop together form an anatomical andphysiological unit.

4. Trituration occurs in the gizzard. The teeth are adaptedto the trituration of plant material; this is of particular impor-tance owing to the weak action of the cellulase.

5. Coarser particles of weed are retained by the teeth of thefilter chamber and returned to the gizzard during the forwardmovement of the gut fluid.' 6. The ciliary currents in the anterior intestine ensure that

only food in a finely divided or fluid state is admitted to thestomach. Medium and larger sized particles are carried straightinto the intestine.

7. Ciliary currents in the stomach are concerned with theremoval of material rejected from the tubules of the digestivediverticula. This material is consolidated, cemented, andmoulded into a faecal rod within the caecum, and conveyed byciliary action to the intestine.

8. The intestine is concerned with the further consolidationand moulding of the complete faecal mass, and its propulsion(by combined ciliary and muscular action) to the rectum.

9. Mucus is secreted throughout the gut with the exception ofthe regions of the jaws, gizzard, and filter chamber. Enzymesare secreted in the salivary glands (amylase and protease) and inthe digestive diverticula (carbohydrases, lipase, and proteases).Glands probably secreting a lubricant (other than mucus) occurin the epithelium of the lateral walls of the buccal cavity, andothers, secreting a cementing substance, in the caecum andintestine.

10. Absorptive cells occupy the greater part of the epitheliumof the digestive diverticula. They occur together with secretory,excretory, and storage cells.

396 H. H. HOWBLIiS

11. Digestion occurs within the oesophagus and crop, gizzard,filter chamber, anterior intestine, stomach, and tubules of thedigestive diverticula. The hydrogen ion concentration is heresuitable for the action of the enzymes, and the gut fluid is keptin motion by the muscular activity of the walls.

12. A high pH exists in the lumen of the caecum, posteriorintestine, and rectum, probably assisting in the consolidation ofthe faecal mass by increasing the viscosity of the mucus.

13. The presence of a highly efficient mechanism for theformation of the faeces is probably correlated with the poorlydeveloped cleansing mechanism in the mantle cavity.

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