The Histology of the Alimentary Tract of the Plaice (Pleuronectes

The Histology of the Alimentary Tract of thePlaice (Pleuronectes platessa).

By

Ben Dawes, A.R.C.S., D.I.C.,

Research Assistant to the Marino Biological Association at Plymouth.

With 12 Text-Bgures.

CONTENTS.P A G E

I N T R O D U C T I O N , M A T E R I A L , A N D M E T H O D S . . . . . 2 4 3

HISTORICAL . . 244

G e n e r a l M o r p h o l o g i c a l N o t e s . . . . . . . 2 4 6

H i s t o l o g y 2 4 8

B u c c a l C a v i t y a n d P h a r y n x . . . . . . 2 4 8

O e s o p h a g u s . . . . . . . . . 2 5 2

S t o m a c h 2 5 4

D u o d e n u m a n d I n t e s t i n e . . . . . . . 2 5 9

P y l o r i c C a e c a 2 6 2

R e c t u m 2 6 4

D I S C U S S I O N 2 6 6

A C K N O W L E D G E M E N T 2 7 2

SUMMARY 272

LITERATURE LIST 273

INTRODUCTION, MATERIAL, AND METHODS.

THE research work about to be described was undertaken asa preliminary to a study of the physiology of digestion in thefish named. Histological examination of tissues must neces-sarily precede a study of the functions of these tissues, especiallyso where one of the functions happens to be secretory. This isnot realized as fully as one would incline to believe, and publishedaccounts of the physiology of digestion of fishes are met withwhere the histology of the alimentary tract of the particularfishes is at present unknown.

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Histological work calls emphatically for freshly preservedmaterial, particularly in the case of a fish. Materials landed as' trawled fish ' in the economic sense are of little or of no use.The fish used in the work to be described were freshly trawled inCawsand Bay and were either brought directly into the labora-tory in the living condition or placed in the constructed ' pond 'at Pier Cellars, into which each tide flows, to be taken out alivewhen required. At the time of fixation, therefore, all tissueswere in the freshest condition.

Bouin's fluid was used exclusively and the state of fixationwas generally very good. Xylol or clove oil or both served asclearing agents and the usual method of paraffin-wax embeddingwas employed. Slides Avere treated with soap and water,chromic acid, and 70 per cent, alcohol with a little added am-monia in turn, and were stored in 70 per cent, alcohol. Sectionswere fixed by the egg-albumen and glycerine method afterstretching on a water bath.

The stains used were Mallory's triple, Delafield's and Ehrlich'shaemotoxylin, mucicarmine, and borax-carmine with picro-nigrosin as counterstain. Two at least, often three, of thesestains were used on each piece of tissue sectioned with a viewto verifying the presence of structures within the cells.

Tissues were taken at various stages during the digestiveprocesses, so as to render clearly the histological changes con-sequent upon digestion. This was made possible by thefacilities to hand for maintaining fish alive.

HISTORICAL.

It is quite impossible in a short historical account to dojustice to the work that has been carried out on the structureof the digestive tract of fishes. The workers have been numerous,particularly during the middle and latter parts of last century.Macroscopic researches commenced about the beginning of thecentury, or rather earlier, when Monro (1795), Home Everade(1814), Eathke (1824), and others all contributed substantiallyto current knowledge of the gross structures of the alimentarysystem.

ALIMENTARY TRACT OF PLAICE 245

With the institution of the microscope came the work ofEathke (1841) on A m p h i o x u s , and that of Miiller (J.)(1843) on M y x i n e , to inaugurate another and more completeseries of researches on the microscopical structure of what hadhitherto been conceived of as gross structures. Agassiz andVogt (1845) prepared sections of the stomach of the trout andobserved that what have been termed ' anfractuosites de lamuqueuse ' appeared as a ' rouleau ' in the form of a club andconsisted of two types of' cellules ', round and cylindrical ones.

Without in the slightest intending to disparage the sincereand careful endeavours of these early workers, one must observethat their attempts did not invariably meet with success.Valatour (1861), in his researches on the teleost fishes, failed torealize the importance of using fresh material, with the resultthat his work was to some extent disregarded, even discredited,by some of his immediate successors. Many of his findingsreceived support, however, when the work of Edinger waspublished. Edinger (1877) enjoyed many distinct advantages,the refinement of histological technique through sixteen years,and the examples and results of several workers on mammalianhistology, men such as Heidenhain (1870) and Eollett (1871).He observed that the gastric glands of fishes differed histologi-cally from those of mammals, showed that the structure ofthe pyloric caeca was very similar to that of the intestine, andremarked upon the absence of intestinal glands.

Macallum (1884) studied the alimentary canal of the ' ganoids',in which he noted and described a ciliated epithelium in theoesophagus and stomach. Pilliet (1894) concerned himselfprincipally with the Pleuronectidae, on which family nothingrelevant to this piece of research has appeared in the literaturesince, excepting the work of Cole and Johnstone (1901). Thework of Pilliet will be mentioned and discussed in the moreappropriate part of this paper. Yung (1899) gave a good accountof the work previously carried out but overlooked that ofGulland (1898), which was probably in the press at his time ofwriting, and merely mentioned that of Stirling (1884). Thelatter worked on the herring, Gulland on the salmon, where

246 BEN DAWES

changes in the nature of the mucosa during spawning migrationswere specially treated.

During the current century a set of three researches are onrecord : that of Sullivan (1907) on Elasmobranchs, recordinga ciliated oesophageal epithelium, that of Greene on the king-salmon (1912), and a short paper by Vonk (1928), which islargely physiological. Thus, from the point of view of moderntechnique, the literature is by no means extensive.

Genera l Morpho log ica l N o t e s .

The alimentary tract of the plaice is a fairly uniform tubewhich, in s i t u , is formed into a number of rather complicatedfolds. It is externally only partially differentiated into regionsof which the following will be recognized in addition to thebuccal cavity: pharynx, oesophagus, stomach, duodenum,intestine, and rectum. The tissue referred to as the pharynxis that situated between and slightly behind the pharyngealteeth, the oesophagus being the posterior continuation of thistissue into the stomach. There is no external indication of thepassage of the oesophagus into the stomach but if the alimentarytract is opened and the mucosa examined in a fresh condition,the line of demarcation is quite strongly shown, the gastricmucosal folds appearing distinctly different from the oesophagealones, even to the naked eye. This line can be taken as beingvery slightly posterior to the posterior wall of the pericardium.That there is sufficient justification for segregating the pharynxand oesophagus will be evident when the histological differenceshave been described.

The oesophagus widens as it passes into the stomach and thelatter organ attains its greatest diameter about the middle ofits length, posterior to which it becomes more slender especiallynear the pyloric sphincter and valve. The free end of this valveprojects into the intestine of course.

Three regions are recognized in the post-pyloric intestine, theduodenum, intestine, and rectum. The first two, which togetherform approximately half the entire length of the alimentarytract, are arbitrary divisions of the pre-rectal intestine, made


with a view to investigating separately anterior and posteriorhalves of this relatively long stretch of gut. The third region,the rectum, is relatively short, but the boundary between itand the intestine is a real one, as will be seen later.

Into the duodenum open the four very small and rudimentary(or vestigial) pyloric caeca and also the bile duct. Along thewhole length of the duodenum and of the intestine, the ultimatefactors of the portal vein, which penetrate the wall of the alimen-tary tract at indeterminate points, are embedded in looseglandular tissue. Similar tissue is found on the mesenteriescarrying these blood-vessels and around the blood-vessels inthe tissue of the liver. It represents the diffuse type of pancreas,which characterizes the teleost fish.

In the empty condition the rectum is not easily recognizedas a separate region, but in the full condition towards the endof a meal it often appears very considerably distended, eventhough the intestine is completely empty. This fact immediatelysuggests the presence of a valve separating the two regions andexamination proves that this is the case. The valve, the freeend of which projects posteriorly, is quite as well developed asis the pyloric valve. It will be referred to as the intestino-rectal valve.

Although the relative lengths of the above-mentioned regionsare doubtlessly subject to considerable variation, the followingcarefully made measurements of the series in a single fish,29-0 cm. long, will impart some idea of their order :

cm.Length of whole tract, uncoiled, from lips to anus . 28-0Lips to anterior margin of pharyngeal teeth (= buccal

cavity) . . . . . . . . . 4-0Anterior margin of pharyngeal teeth to anterior end of

stomach (= pharynx plus oesophagus) . . . 2 - 8Anterior end of stomach to pyloric valve . . . 4 - 7Pyloric valve to intestino-rectal valve (= duodenum plus

intestine) . . . . . . . . 1 4 - 5Intestino-rectal valve to anus (= rectum contracted) . 2-0

248 BEN DAWBS

H i s t o l o g y .

The entire alimentary tract, with the exception of the pharynx,consists of the following layers commencing from without andproceeding towards the lumen :

(1) Serosa : a thin layer of areolar tissue on which connectivetissue-cells are superimposed.

(2) Outer longitudinal muscular layer : a thin sheet of muscle-bundles having longitudinally disposed fibres.

(3) Inner circular muscular layer : a relatively thicker sheetof transversely disposed fibres.

(4) Sub-mucous coat: a layer of more or less loose areolarconnective tissue in which are embedded numeroussmall blood-vessels and fine nerve-fibres.

(5) Mucous membrane: a membrane of variable thicknessaccording to the region to which it belongs and to thedegree of distention of that region during digestion.

These layers, therefore, characterize the fish. They vary incertain features all along the tract. The muscular layers andthe mucous membrane show the greatest histological variationregionally, the serosa and the sub-mucous coat being fairly con-stant in histological detail throughout. There is no trace of amuscularis mucosa, a stratum granulosum, or a stratum com-pactum in any part of the tract. In the description whichfollows, wearying repetition of names will be avoided as muchas possible, mention being made chiefly of the histologicalvariations met with in the regions included between the lipsand the anus.

Bucca l Cav i ty and P h a r y n x .

The mucous membrane of the buccal cavity consists of astratified epithelium which rests upon a basement membrane.Below it is a layer of loose areolar tissue containing minuteblood-vessels. The epithelium is gently corrugated, the foldsrunning longitudinally and containing short processes of areolartissue. In addition to these corrugations, microscopic papillaeof connective tissue project into the epithelium at certain


indeterminate points (Text-fig. 1, p.a.t.), which papillae eachcontain small blood-vessels and in some cases a fine nerve-fibrilalso. The epithelium is about ten to twelve cells in thickness,the deeper cells being columnar with rounded extremities, whilethe superficial cells are polyhedral or round. Apparently thereare no scale-like cells at or near the periphery. The nuclei aregenerally spherical or, in the basal cells, oval. Minute inter-cellular spaces appear to exist between the epithelial cells.

TEXT-FIG. 1.

Vertical section of the mucous membrane of the buccal cavity, a.t.l.,areolar tissue-layer ; bl.v., blood-vessel; ep., epithelial layer ;n.ep., nuclei of the epithelial cells ; p.a.t., papilla of areolartissue passing into the epithelium. ( x 150.)

Goblet-cells are exceedingly rare, but some few are found asperfectly isolated units. These are spherical or oval in form,appear to lack the tail-like process usually found- at the baseof goblet-cells, and they open to the exterior by a relatively widepore. Structures resembling the taste-buds of mammals occurbut extremely rarely. These are a characteristic feature of themucous membrane of thepharynx (vide infra), and for allpractical purposes they can be neglected in the case of the buccalcavity.

Considerable differences are seen in the pharynx. The epi-thelium, like that of the buccal cavity, is stratified but consistsof fewer cell-layers (Text-fig. 2, ep.c). It is intensely folded sothat the lumen is reduced to a series of much-branched canals

250 BEN DAWES

in the contracted condition. The main lumen is preserved,however, by virtue of the fact that the most superficial foldsare extensive transversely. Finger-like processes of the areolartissue-layer project into the folds no matter how minute these

TEXT-FIG. 2.

t bp

Transverse section passing through a mucosal fold of the pharynx.Areolar tissue-layer rendered in black, ep.c, epithelial cells ; g.c,goblet-cells ; n.ep., indeterminate nuclei of epithelial cells rich inchromatin ; o.g.c, openings of goblet-cells ; t.b., ' taste-bud ' ;t.b.p., shallow pit in the epithelium seen at the periphery of thetaste-bud. ( x 200.)

happen to be. Goblet-cells are extraordinarily numerous,occurring in an unbroken sequence throughout the extent ofthe rnucosa, so that one never succeeds in locating an area devoidof them, or even where the regular sequence is broken. Theyare most numerous in the deeper folds, being two, three, or moredeep, while in the superficial folds which bound the lumen theyusually form shallow patches one, or at most two, goblets deep(Text-fig. 2, g.c). In any situation they are not found typically


at the surface but usually lie more deeply situated, the gobletcommunicating with the lumen of the pharynx by a conspicuousneck and a small pore. The goblets are oval except wherelaterally compressed due to great crowding and where underlyingother goblet-cells, when they are spherical. The basal processis not filamentous, but appears like a small accretion on the baseof the goblet. The stratified nature of the epithelium probablyaccounts for the development of this unusual type of cell.

Near the base of the pharyngeal epithelium in the superficialfolds bounding the lumen cells are found, the nuclei of whichshow conspicuous heavily stained chromatin masses. They areless conspicuous in the deeper folds of the mucosa, where theepithelium is thinner and composed of fewer cells, despite thefact that goblet-cells are more numerous. These appear to beactively dividing cells of the epithelium (Text-fig. 2, n.ep.).

In the peripheral part of the epithelium of the pharynx, andespecially in the superficial folds bounding the main portion ofthe lumen, small clusters of cells resembling taste-buds occur.The individual cells of each cluster are of an elongate spindle-shape, possess oval nuclei, and are closely packed together(Text-fig. 2, t.b.). The spindle-cells reach almost to the basementmembrane, from which they are separated by columnar cells.Towards the periphery they terminate each in a fine, deeplystaining process, which projects into a pit-like depression in themucosa (Text-fig. 2, t.b.p.). The spindle-cells, which on analogywith similar cells in the mammal, can be regarded as gustatorycells, do not appear to possess branched basal processes such asare found in some cells of this type.

The musculature of the pharynx consists largely of a thickcircularly disposed band of striated fibres, which is continuouswith the circular musculature of the oesophagus and stomach.In addition to this, there exist a number of loosely arrangedmuscle-bundles, longitudinally disposed but slightly oblique,which form a second series within the areolar tissue-layer. Thesebundles also consist of striated fibres. There is no continuumof the outer longitudinal musculature of the oesophagus andstomach in this region.

'252 BEN DAWES

O e s o p h a g u s .The serosa is an extremely thin layer of areolar connective

tissue continued externally into the tissue of the mesenteries andinternally into the connective tissue binding together the muscle-bundles immediately beneath it (Text-fig. 3, ser.). It is pene-trated at indeterminate points by small blood-vessels and finenerves as these enter the tissue of the alimentary tract. Theouter longitudinal musculature does not take the form of anunbroken ring, being represented by irregularly disposedbundles (Text-fig. 3, l.m.l.), while the inner circular muscularlayer is of enormous thickness, at least ten times the thicknessof the inner layer (Text-fig. 3, c.m.l.). All muscle-fibres are of thestriated type, as in the pharynx. The loosely arranged muscle-bundles observed in the areolar tissue-layer of the pharynx arecontinued throughout the oesophageal region as even morescattered bundles forming crescentic masses opposite themesenteries (Text-fig. 3, m.b.).

The mucosa is folded to an even greater degree than in thepharynx, or incidentally in any other part of the tract. Thefolds form primary and secondary series as regards branching,and transverse sections of this region show a maze of foldedmucosa and occluded lumen. A small and relatively simple foldis shown in Text-fig. 3. Wider channels of lumen remaincentrally to form a stellate canal into which the smaller channelsof lumen open. The epithelium is no longer stratified but whereunmodified by the presence of goblet-cells consists of a singlelayer of columnar, but not very tall, cells with oval nuclei whichtake up the major part of the volume of their cells. These un-modified regions are to be found at the tips of the branches ofthe mucosa (Text-fig. 3, t.m.f.). Goblet-cells are numerous butoccupy only the depressions of the mucosa, where they formextensive patches which contrast strongly with the unmodifiedpapilla-like tips. They form only a single series and are usuallylaterally compressed due to crowding. The nuclei are to befound embedded in a crescentic mass of cytoplasm at the base ofthe goblets. Below the goblet-cells is a ' cushion ' of smallrounded cells, the only suggestion of stratification in the


epithelium. With the fixative employed, the contents of thegoblets present a reticular appearance.

Although occasional leucocytes are found between the un-

TEXT-FIG. 3.

Transverse section of a portion of the oesophagus, showing theintense degree of muoosal folding, a.t.l., areolar tissue-layer ;c.m.l., circular muscular layer; g.c, goblet-cells of the epitheliallayer; l.m.l., longitudinal muscular layer ; rn.b., loosely arranged,longitudinally disposed muscle-bundles within the areolar tissue-layer ; ser., serosa ; t.m.f., tips of the mucosal folds lackinggoblet-cells. (x40.)

modified cells of the epithelium, it seems unlikely that suchisolated units can materially aid food absorption. The histologi-

254 BEN DAWBS

cal features enumerated above for the pharynx and oesophagus,the intensely folded mucosa, well-developed striated muscula-ture, abundance of mucous-producing cells, tend to demonstratethat this part of the alimentary tract is principally, if not solely,

TEXT-FIG. 4.

Longitudinal section through the junction of oesophagus andstomach. The arrow shows approximately the line of demarca-tion, oesophagus above, stomach below, a.t.l., areolar tissue-layer ; c.m.l., circular muscular layer ; g.c, goblet-cells ; g.cr.,crypt into which gastric gland opens ; g.gl-, gastric gland ; oe.ep.,oesophageal epithelium; s.g.ep., superficial epithelium of thestomach. ( x 50.)

concerned with a facile swallowing of large morsels of food,which may be selected by means of the gustatory organs.

S t o m a c h .

Although, as has been stated, there is no external morphologi-cal indication of the passage of the oesophagus into the stomach,yet there is a fairly sharp line of demarcation between the organshistologically. The transition from oesophagus to stomach ismarked first of all by the loss of the complex mucosal folds,in place of which are seen in the stomach seven or eight largeand more or less equal longitudinal folds. In the contractedcondition of the organ, these are approximated so that the lumen


is resolved into seven or eight radiating channels of narrowcalibre (Text-fig. 5, l.st.). It is also marked by the disappearanceof goblet-cells (Text-fig. 4, s.g.ep.). A longitudinal section showsthe abrupt nature of the change and also that the oesophageal

TEXT-FIG. 5.

bL v

Diagrammatic transverse section of the stomach in the empty condi-tion, a.t.l., areolar tissue-layer ; bl.v., blood-vessel; c.m.l., circularmuscular layer; g.p.m., deep, glandular part of the mucosa ;l.m.l., longitudinal muscular layer; l.st., lumen of stomach;o.p.m,, superficial part of mucosa containing crypts; ser., serosa.(x25.)

epithelium is continued into the superficial epithelium of thestomach (Text-fig. 4, oe.ep., s.g.ep.).

The gastric mucosa varies in thickness in different parts of theorgan, being much thicker in the middle and posterior parts thananteriorly. The thickness is directly clue to the degree of folding,i. e. to the degree of development of the gastric glands. Theseare both deeper and more numerous in the mid-stomach andposteriorly. Anteriorly, they are very shallow (Text-fig. 6),although the crypts are well marked, and here resemble oeso-

256 BEN DA WES

phageal folds (Text-fig. 6, g.cr.). The glands are of the simpletubular type, but appear to be slightly branched in the mid-stomach and posteriorly, and are closely aggregated. The super-ficial epithelium is formed into a series of pit-like depressions

TEXT-FIG. 6.

•gcr

•upep

Transverse section passing through a mucosal fold near the anteriorextremity of the stomach. Indicating the shallow and simplenature of the gastric glands, a.t.l., areolar tissue; g.c, goblet-cells;g.cr., crypt leading into lumen of gland ; gran.o., granular cells atbase of gland tubule ; rawcc, mucus-producing cells forming theneck of the tubule ; sup. ep., superficial epithelium. ( x 125.)

or crypts which lead into the glands. This superficial epitheliumconsists of columnar cells rather taller than those of the un-modified tips of the mucosal folds in the oesophagus. The nucleiare oval and difficult to stain during a prolonged resting period,e. g. during the period of the ' empty stomach '. The cellcytoplasm behaves in a striking manner to stains. Near theperiphery is a narrow blue-stained zone (with Mallory); rather


more deeply situated is an equally narrow red-stained zone, andin the middle region of the cell just outside the nucleus is a broad

TKXT-FIG. 7.

sup ep

Vertical section of a gastric gland from the mid-stomach, a.t.l.,areolar tissue; bl.c. and bl.v., blood-corpuscles and vessel ;g.cr., crypt (rendered in perspective as seen in a relatively thicksection); gran.c, granular cells ; l.gl., lumen of gland ; muc.c,mucus-producing cells; sup.ep., superficial epithelium liningcrypt. ( x 300.)

blue-stained mass. This latter is also visible with borax-carmine, picro-nigrosin staining as a greyish mass. With

NO. 290 S

258 BEN DAWES

mucicarmine one obtains no stain except in the peripheral partof the cells lining the crypts. These superficial cells are probablyslightly mucus-producing, but are not typically so.

The gastric glands consist of cubical cells of two types. Inthe neck of the glands immediately below the crypts occur cellswhich, during a prolonged resting phase, appear devoid ofgranules and unstained or faintly blue with Mallory or Dela-field's haematoxylin. During active digestion these cells showonly a border behaving in the above manner, the basal part ofthe cell staining more deeply. With mucicarmine these cellsstain reddish, so that they are undoubtedly mucus-producingcells. They can be compared to the mucoid cells scatteredamong the so-called peptic cells of the mammalian fundic gland.The basal part of the gland tubule consists of granular cells,which are probably the enzyme-secreting cells of the gland.There is no sign of cells resembling the oxyntic or parietal cellsof mammals in any part of the gastric mucosa. The componentcells of the glands are illustrated in Text-fig. 7, sup.ep., muc.c,gran.c.

Text-fig. 8 illustrates diagrammatically a hypothetical conception of the possible transition from the oesophageal type ofmucosa to the gastric type. The intermediate type does notexist as shown in B, but an approximation to it is seen at thejunction of oesophagus and stomach (Text-fig. 6, bottom, right).The homologous tissues are : (1) the tips of the oesophagealmucosal folds and the superficial epithelium of the stomach ;(2) the goblet-cells of the oesophagus and the mucus-producingcells of the gastric tubule ; and (3) the cushion of cells below thegoblet-cells in the oesophagus and the granular basal cells ofthe gastric gland.

Near the pyloric sphincter the gastric glands become moreshallow and opposite the sphincter, they disappear altogetherwhile the crypts remain (Text-fig. 11, sup.ep.). The sphincteris merely the enormously thickened circular musculature. Themuscles of the stomach consist of non-striated fibres, the transi-tion from the striated type occurring near the anterior end ofthe organ. Here also the loosely arranged muscle-bundles in

ALIMENTARY TRACT OP PLAICE 259

the areolar tissue-layer disappear. In the mid-stomach theinner and outer muscular layers are of approximately the samethickness, the former gradually thickening near the pyloricsphincter. The pyloric valve contains muscles from both theabove layers but largely from the inner one. Goblet-cells occur oneach side of the valve although the types of mucosal folds differ,

A S CDiagram illustrating an hypothetical conception of the transition

from the oesophageal type of mucosa to the gastric type. A,oesophagus ; C, stomach, and B, hypothetical intermediate type.a.l., areolar tissue ; ep., in A, epithelium of tips of mucosal foldspassing through B to form the superficial epithelium continuedinto the crypts in C ; m.c, in A, goblet-cells forming the mucus-producing part of the gland tubule in C ; gl-c, in A, the ' cushion 'of epithelial tissue beneath the goblet-cells in the mucosal troughs,in C, the granular cells at the bases of the tubules.

those on the anterior aspect to some extent resembling gastriccrypts.

D u o d e n u m and I n t e s t i n e .

The mucosa of the duodenum and intestine is relativelysimple in structure. In bulk it is gently corrugated into a largenumber of longitudinal folds which, unlike those of the oeso-phageal mucosa, are unbranched. During the period when thetract is empty, slight transverse folds also occur. The folds t,osome extent simulate villi, while the mucosal depressions give

S2

260 BEN DAWES

a false impression of crypt-like glands, especially in materialfixed during a resting phase when the organ is empty. Duringdigestion the intestine is greatly distended and the folds smoothedout. That villi and glands do not occur is indicated by the lackof histological differentiation in the mucosa, which consists of

TEXT-FIG. 9.

epc S m

Transverse section of a mucosal fold in the duodenum, fixed duringa prolonged, resting phase. Areolar tissue rendered in black.b.g.c, filamentous basal part of the goblet-cells ; ep.c, columnarcells of epithelium; g.c, goblet-cells; I.e., leucocytes; n.z.,spindle-shaped zone containing nucleus ; s.m., darkly stainingborder of epithelium. (x250.)

tall and extremely slender columnar cells interspersed withgoblet-cells. The nuclei are small and oval and, like the nucleiof the cells forming the gastric tubules, are difficult to stainduring a prolonged resting phase, when the cytoplasm stainsmore deeply. Tissue fixed at the end of such a phase in Bouin's


fluid and stained in section Avith Delafield's haemotoxylin showsa deep-blue spindle-shaped mass about three times the lengthof the nucleus situated about the middle of the cell. In materialfixed during active digestion the only parts of the nucleus takingup stain are the periphery and a fine chromatin network. Theouter half of each cell also stains less deeply and the margininstead of appearing heavily stained is now quite pale. At thesame time a rather darker mass appears just below the pale

TEXT-FIG. 10.

•p s m

Vertical section of the intestinal mucosa, fixed during active diges-tion of food, a.t., areolar tissue; d.s.c, rather more deeplystaining cytoplasm ; ep.c, columnar cells of epithelium ; I.e.,leucocytes ; n.ep., nuclei of epithelial cells ; p.s.m., margin ofepithelium, now pale-staining. ( x500.)

border. Text-fig. 9 shows the epithelium during a resting phase,Text-fig. 10 during active digestion.

Numerous isolated goblet-cells occur in the epithelium. Theyare not found in patches as in the oesophagus and often rela-tively large areas of mucosa are seen to be devoid of them. Theydiffer from the corresponding cells in the pharynx and oeso-phagus in that they are smaller, regularly oval, and possessa filamentous basal portion which contains the nucleus (Text-fig. 9, g.c, b.g.c).

Leucocytes are to be found between the epithelial cells of bothduodenum and intestine at all times. Their small roundednuclei, which stain blackish with Delafield's haemotoxylin,render them conspicuous (Text-fig. 9 and Text-fig. 10, Lc).

262 BEN DAWES

They appear in all zones to the margin of the epithelium, whichapparently they never penetrate. If anything they appear inslightly greater numbers during digestion, although there is nomarked change and it appears unlikely that they play anyconsiderable part in this process.

Processes of the areolar tissue-layer pass into the mucosalfolds and carry small blood-vessels. There is no sign of a centrallacteal, so that the folds are definitely not villi. In view of thefact that the muscularis mucosa is absent, lacteals homologouswith those of mammals could not exist in the plaice, since thislayer in part forms the wall of these structures. It is probablethat the spaces in the areolar tissue serve as lacteals in this fish.

The musculature of the duodenum and intestine consists ofequally thick circular and longitudinal layers. The fibres areof the non-striated type of course. Between the two layers occurthe nerve-fibres and cell-clusters of the plexus of Auerbach,Avhich appears very prominently in all sections of these regions.In material treated by the usual methods of histological tech-nique there is no sign of a ganglionated plexus comparable withthat of Meissner, which is found in the sub-mucous coat of themammalian intestine.

The serosa of the intestine is much thicker than is that ofmore anteriorly situated regions of the alimentary tract.

P y l o r i c Caeca .The histological structure of the pyloric caeca is similar to

that of the intestine. The muscular layers are thinner and themucosa is rather more considerably folded, although the foldsare simple like those of the duodenum and intestine. Theincreased degree of folding appears to be due entirely to thealmost spherical form of the caeca, which, as has already beenstated, are very small. The lumen of each caecum is necessarilyreduced but by no means obliterated, so that it communicatesfreely with the lumen of the duodenum (Text-fig. 11, l.py.c).Thus the caeca are practically pockets of the intestine into whichfood can readily pass.

The epithelium is similar to that of the duodenum, consisting

ALIMENTARY TRACT OF PLAICE

TEXT-FIG. 11.

263

•a t L

Lpyc

Longitudinal section passing through the pylorus and a pyloriccaecum, a.t.l., areolar tissue-layer; c.m.L, circular muscular layer;ep.py.c, epithelium of caecum; l.m.l., longitudinal muscularlayer ; l.py.c, lumen of caecum ; p.sph., pyloric sphincter ; pi. ofA, nerve-fibres of Auerbach's plexus; py.v., pyloric valve; ser.,serosa; sup.ep., superficial epithelium of stomach (note theabsence of glands in this region); v.m., process of circular musclescontributing to the pyloric valve. ( x 30.)

264 BEN DAWES

of tall and slender columnar cells interspersed with goblet-cells.Leucocytes are found between the columnar cells just as com-monly as is the case in the duodenum and intestine. Betweenthe muscular layers the plexus of Auerbach is conspicuous also(Text-fig. 11, pi. of A). In fact all the characteristic features ofthe post-pyloric intestine are repeated in the structure of thecaeca, which can thus be regarded as an integral part of thealimentary tract in this region.

E e c t u m .

The boundary between the intestine and the rectum is markedby the intestino-rectal valve, as was stated earlier. This isquite as well developed as the pyloric valve which marks theboundary between the stomach and the duodenum. It differsfrom this latter valve in that its musculature is derived equallyfrom that of.the tract on each side of it (Text-fig. 12, m. of v.).The circular muscles of both intestine and rectum becomeslightly thicker near the point of origin of the valve and passentirely into it, accompanied by a process of the longitudinalmuscular layer. The nerve-fibres of Auerbach's plexus also passinto the tissue of the valve. The mucosa is of the intestinaltype on both faces (Text-fig. 12, ep.int., ep.rect.), and there aremore goblet-cells on the rectal face. The free end of the valveprojects posteriorly.

The mucosa of the rectum is similar to that of the intestine,the chief differences being a greater degree of folding in theclosed condition and more numerous goblet-cells. These latterform an almost continuous sequence in some areas of tract andare of the same type as the corresponding cells of the intestine.Leucocytes occur between the columnar cells of the epitheliumalmost as commonly as in the intestine and at all times.

The musculature of the rectum is much thicker than that ofthe preceding part of the alimentary tract. The inner, circularlayer is more than twice as thick, the outer, longitudinal layeralmost twice as thick as the corresponding layers in the intestine.The inner layer becomes suddenly thickened near the anus,

ALIMBNTABY TRACT OP PLAICE

TEXT-FIG. 12.

265

Ser

pL oFk

m of v

Longitudinal section of the junction of intestine and rectum, a.t.l.,areolor tissue-layer ; bl.v., blood-vessel; c.in.l., circular muscula-ture ; ep.int., intestinal epithelium ; ep.rect., rectal epithelium ;i.r.v., intestino-rectal valve ; 1.int., lumen of intestine ; l.in.l.,longitudinal musculature ; l.rect., lumen of rectum ; m. of v.,musculature of valve; pi. of A, Auerbach's plexus ; ser.,serosa. ( x40.)

where it forms the anal sphincter. Here, the mucosal folds arevery numerous and very deep, while the goblet-cells completelydisappear.

266 BEN DAWBS

DISCUSSION.

When a consideration is made of the structure of the alimentary tract in the various groups of fishes, many histologicaldifferences are found. The majority of these are bound up withgross morphological differences such as the types of stomach,the presence or absence of pyloric caeca or of a spiral intestinalvalve and others. But differences occur apart from these.Macallum (1886) observed a ciliated epithelium in the oesophagusof Arnia, L e p i d o s t e u s , and A c i p e n s e r , and even inthe stomach of the first named. Sullivan (1907) describes asimilar epithelium in the oesophagus of the Elasmobranchs. Itappears that such an epithelial layer does not occur at all in theTeleostei as neither Greene (1912) nor Stirling (1884) observedit in the king-salmon and herring respectively. Gulland (1898)unfortunately omits a consideration of this region in the salmon,but in the plaice the present research shows that cilia are absentfrom every part of the tract. Nor did Pilliet (1894) observe aciliated epithelium in any of the Pleuronectidae he studied.

Although there is no external indication of the passage of theoesophagus into the stomach in the plaice, there is, as we haveseen, a definite, sharply defined histological boundary betweenthe two regions. The mucosa of the oesophagus with its much-branched and complicated longitudinal folds and abundance ofgoblet-cells passes abruptly into a glandular stomach devoid ofsuch folds and cells. Cole and Johnstone err in remarking thatthe oesophagus is sharply defined from the stomach by thestrongly developed transverse musculature at the proximal endof the latter region. On the contrary, the immense circularmuscles of the oesophagus diminish in thickness gradually inpassing towards the mid-stomach. There is no abrupt change inthickness at any point. In passing it might also be added thatthe feebly developed longitudinal musculature consists ofstriated fibres and not of non-striated elements, as these writersmaintain. They did not observe the loosely arranged muscle-bundles in the areolar tissue-layer.

The oesophagus of the herring is abundantly beset with goblet-


cells, between the upper ends of which Stirling also found smalltriangular cells intercalated. Greene did not observe goblet-cells in the king-salmon but instead found cells with basal nucleiand clear outer margins, which he regards as mucus-producingcells. Goblet-cells are very numerous in the oesophagus of theplaice, but there is no sign of an intercalated series of triangularcells, although occasional cells are found in this situation. Theseare explained when it is considered that cells below the peripherydevelop into goblet-cells in the depths of the folds where theepithelium is to some extent stratified. Unmodified cells,therefore, can remain in a superficial position and in otherpositions without forming a definite layer.

Only a few workers have differentiated between the pharynxand the oesophagus, probably because the two regions togetherform only a relatively short length of tract. Three featuresjustify one in considering the pharynx as a definite region: theabsence of longitudinal musculature, the definitely stratifiedepithelium with its abundance of goblet-cells, and the presenceof structures referred to as taste-buds. I have not met with anyhitherto published account of the occurrence of taste-buds inthe pharynx of the teleost fish, but Macallum (1886) describessuch structures in the oesophageal mucosa of A c i p e n s e r ,remarking on the rare occurrence of these in a similar situationin the higher vertebrates. This leads him to the conclusionthat the piscine and mammalian oesophageal regions are nothomologous. Comparison of these regions in the plaice and themammal is not particularly yielding, but it is conceivable thatthe deep folding of a mucosa richly supplied with goblet-cellsmight lead to the development of definite mucous glands in thefolds, thus removing the necessity for superficial goblet-cells.If mucous glands of mammals have developed in this way, thenthe pharynx of the fish, possessing taste-buds as well as incipientmucous glands, would correspond to the similarly named regionin the mammal plus adjacent regions such as the epiglottis andthe tongue, i.e. the structures bearing the gustatory organs.

From the description given, it seems clear that the functionsof the oesophagus and pharynx are gustatory, mucus-producing,

268 BEN DAWBS

and the facilitating of rapid and efficient swallowing action. Theabsence of definite glands and specialized cells suggests thatthese regions possess little or no secretory power apart from thatof mucus-production.

Heidenhain (1870) discovered two kinds of cells in the tubulesof the gastric glands of mammals. These are now referred toas central and parietal or oxyntic cells. Edinger (1877) madea detailed study of the histology of the digestive tract of fishesand found that only one type of cell occurs in the tubules andthat this type was neither central nor parietal. Gulland (1898)recognized two kinds of epithelium in the gland tubule of thesalmon, but agreed that the parietal type of cell is non-existent.The tAvo epithelial types he refers to as intermediate and zymin-producing, the former being found in the neck of the gland whilethe latter forms the base of the tubule. He remarked on theappearance of a clear outer hem in the intermediate cells.Green (1912) says, ' There is no intermediate structurally inter-mediate type of neck-cell in the king-salmon as suggested byGulland for the Atlantic salmon '. He believes that this typeof cell is merely ' a foreshortened type of cylindrical cell'. Butin the plaice two distinct types of cell occur in the gland tubuleand are most evident during a prolonged resting phase. In theneck of the gland, cells occur which are only faintly stained inordinary preparations but which are undoubtedly stained withmucicarmine, thus being mucus-producing cells. At the base ofthe tubule the cells are very granular and undoubtedly areenzyme-producing cells. The mucous cells are inconspicuous,relatively, during digestion, and it is suggested here that Greeneprobably overlooked them as a result of sectioning material fixedduring digestive phases when they were not evident types.Gulland, being chiefly concerned with the period of the spawningmigration, would use material fixed during a prolonged restingphase when, in the plaice at least, the cells are best demon-strated. Stirling (1884) also observed clear cells apparentlyfilled with mucus in the ducts of the gastric glands of the cardiacsac of the herring, but did not emphasize this finding.

It appears that all workers are agreed that parietal or oxyntic

ALIMENTARY TBACT OF PLAICE 269

cells do not exist in fish, and as these cells are deemed hydro-chloric acid producing the question of the source of the acid isstill unsettled. Stirling, remarking on this fact, conceives itpossible that the superficial cells perform more than onefunction and produce both the acid and mucus as well. In theplaice the cells of the neck of the tubules are undoubtedly theagents responsible for the production of mucus, being possiblyderived from the goblet type of cell. The superficial cells, whichare not typical mucus-producing cells, are probably in someway connected with the production of the acid found in thestomach during digestion. Gulland apparently under-estimatedthe importance of the cells in the neck of the tubule.

In the king-salmon gastric glands do not occur near thepyloric valve. Similarly in the plaice the region opposite thepyloric sphincter is devoid of glands although possessing crypts.According to Macallum, however, in the pyloric region ofA c i p e n s e r , ' the majority of the cells are open peripherally ;from this and from their being swollen they bear a resemblanceto goblet-cells.' Thus, if the fixation of Macallum's materialwas all that could be desired, the absence of goblet-cells fromthe stomach is not general in fishes.

Mention must now be made of the work of Pilliet (1894). Heinvestigated the stomach of the Pleuronectidae, using the turbotchiefly but also making use of the sole, the dab and the plaice.In the last two he was most unfortunate, and referring to thesehe says, ' Enfin le Carrelet nous a offert une exception quimerite d'etre signalee. L'estomac etait vide, et nous avons eubeau multiplier les coupes de la muqueuse apres la fixation parl'alcool, les glandes gastriques nous ont echappe.' And again,' Pour la Limande, la m&me difficulty s'etait presentee, mais nousavions reussi a tomber enfin sur la region glandulaire.' He lateradds that after mounting supposed sections of the stomach ofthe plaice they invariably found that they had missed thestomach region, having obtained sections of either the oeso-phagus or the region of the pylorus. His explanation cannot beoverlooked. He says, ' II faudrait done conclure que les glandesdiminuent de nombre et de volume pendant certaines periodes,

270 BEN DAWBS

pour reprendre champ pendant certaines autres.' This mostdecidedly is not the case in the plaice even during the periodwhen the fish characteristically possesses an ' empty stomach '.In the large number of fishes I have dissected during this andother periods, the stomach is unchanged except for the con-traction naturally consequent upon an empty stomach. It seemsnatural that Pilliet should conclude that ' l'cesophagus n'estpas, comme chez les vertebres superieurs, un conduit bien dis-tinct ; il contribue a former la poche stomachale ', with whichconclusion I must strongly disagree. But he observed that thegastric glands of theturbot did not contain parietal cells, remark-ing that ' on voit seulement celles qui se rapprochent du colde la glande etre a la fois plus refringentes et plus opaques quecelles du fond des cul-de-sac dont le plasma est parseme de finsgranules'. Thus, the neck-cells of Gulland and the correspond-ing cells in the plaice did not escape his notice.

The accounts of other workers on the histology of the intes-tinal part of the alimentary tract do not readily lend themselves

• to discussion. Most investigators agree that it is extremelysimple, consisting, as it does in the plaice, of a folded mucosaformed chiefly of an epithelial layer of columnar cells. Thesecells do not show any pronounced granular structure, nor dothey indicate any differentiation except into goblet-cells. Thefolds to some extent simulate villi and crypts, but that thesestructures do not exist is shown by the absence of lacteals andgranular cells such as those of Paneth. But as regards secretorypower it is observed in the plaice that the nuclei of the epithelialcells are not evident in stained preparations of material fixedduring a resting phase. Instead one sees a spindle-shaped massof much greater length than the nucleus, which during digestionshows up as a small oval body, which occupies the middle of thecell and which stains heavily. In material fixed during digestiononly the boundary of the nucleus and a fine chromatin networkstains. That this phenomenon is connected with secretion I feelfairly certain.

Goblet-cells are not as numerous as in the oesophagus andare of a different type, possessing a filamentous basal portion

ALIMENTARY TEACT OF PLAICE 271

which contains the nucleus. Leucocytes are to be foundbetween the cells of the epithelium at all times and in all zones,but are present in relatively small numbers which appear fairlyconstant. For this reason it appears doubtful that they playany considerable part in the absorption of food materials. Thereare no villi and no lacteals in the areolar tissue below the mucosa.It appears that the products of digestion pass through theepithelium into the reticulum of the. areolar tissue-layer andthus directly into the blood-stream.

What has been maintained for the duodenum and intestinealso holds for the pyloric caeca which are identical in histologicalstructure with these regions, and which are in open communica-tion with the lumen of the duodenum. It has been maintainedthat these are organs for storage of reserve food materials,structures concerned with secretion, with absorption, or withboth these latter functions. In the plaice they are small andrudimentary or vestigial, but they present no differences in anyrespect from the duodenum in histological detail and are un-doubtedly as much concerned with secretion and absorptionas is this latter region.

Cole and Johnstone (1901) state that in the plaice ' there isno essential difference between intestine and rectum, but it isconvenient to distinguish the terminal portion of the alimentarycanal from that immediately preceding it '. From this passageit is clear that they overlooked the presence of an ' intestino-rectal' valve which separates the two regions. This is quite aswell developed as the pyloric valve, from which it differs inderiving its musculature from the muscle-layers on each sideof it and not from that on one side alone. The rectum differsfrom the intestine in being more abundantly supplied withgoblet-cells and in having a thicker musculature. This lattermay be due to a relatively greater degree of contraction followingfixation. The anal sphincter is formed from the circular muscula-ture and is much shorter than the pyloric one. It is curious thatthe final portion of the rectum should be devoid of goblet-cells.

•272 BEN DAWES

ACKNOWLEDGEMENT.

I desire to express my sincere appreciation of the manykindnesses shown me by Dr. E. J. Allen, F.R.S., the Directorof the Marine Biological Laboratory, Plymouth, who has sparedneither encouragement nor assistance when these were needed.The work was carried out upon his suggestion at the CawsandLaboratory of the Marine Biological Association. My thanksare also due to Dr. E. S. Eussell of the Ministry of Agricultureand Fisheries, for his encouragement of the work. I have tothank also the various members of the staff at Plymouth whohave assisted in any way, and the skipper and crew of theS.iS. ' Salpa ', who trawled the fish used and landed them at the.Cawsand Laboratory, often in bad weather, during the winterof 1928.

SUMMARY.

1. Structures similar in detail to the taste-buds of mammalsoccur in the epithelium of the pharynx, which is considered asa distinct region for reasons given.

2. The oesophagus possesses an extremely well-developedmusculature composed of striated fibres and an intensely foldedmucosa liberally beset with goblet-cells. There are no glandspresent, and it is sharply denned from the stomach.

3. The musculature of the stomach consists of non-striatedfibres, and there is no differentiation between the gastric glandsat the ' cardiac ' and ' pyloric ' ends of the stomach, except thatin the former position they are more shallow.

4. Three types of cells enter into the composition of the gastricmucosa, superficial cells forming the surface epithelium andthat lining the crypts, mucus-producing cells forming the neckof the tubule, and granular cells forming the basal, secretoryportion of the tubule. Parietal or oxyntic cells do not occur,nor do goblet-cells, in any part of the gastric mucosa.

5. The intestinal mucosa is folded so as to simulate crypts andvilli, but it is shown that these structures do not occur. Thereis no differentiation of the intestinal epithelium except intogoblet-cells. Leucocytes are seen in all zones of the epithelium


at all times but in relatively small numbers which appear con-stant, so that they probably do not play any considerable partin food absorption.

6. The pyloric caeca exhibit the same histological structureas the intestine and are in open communication with this partof the alimentary tract. They are probably secretory andabsorptive like the duodenum and intestine.

7. A well-developed valve occurs at the junction of the intes-tine and the rectum. The rectum shows the same structure asthe intestine except that the folds are deeper and more consider-ably beset with goblet-cells and the musculature thicker.Goblet-cells disappear from the mncosa near the anus.

LlTEEATUBE LlST.

(1) Agassiz, L., and Vogt, G.—" Anatomie des Salmons ", ' M6m. Soc. sc.nat. Neuchatel', torn, iii, 1845.

(2) Cole, F. J., and Johnstone, J.—" Pleuronectes ", ' L. M. B. C.Memoirs', 8 Dec. 1901.

(3) Edinger, L.—" Ueber die Schleimhaut des Fischdarrnes nebst Bemer-kungen zur Phylogenese der Driisen des Darrnrohres ", ' Archivfiir Mikrosk. Anat.', Bd. xiii, 1877.

(4) Greene, C. W.—" Anatomy and Histology of the Alimentary Tract ofthe King-Salmon ", ' Bull. U.S. Bur. Fish ', 32, Doc. 777, 1912.

(5) Gulland, G. L.—" The Minute Structure of the Digestive Tract ofthe Salmon, and the Changes which occur in it in Fresh-water ",' Report of Investigations on the Life-history of the Salmon inFresh-water', Ed. D. Noel Paton, Fishery Board for Scotland(Salmon Fisheries), 1898.

(6) Heidenhain.—" Untersuchungen iiber den Bau der Labdriisen ",' Arch, fiir Mikrosk. Anat.', Bd. vi, 1870.

(7) Home Everard.—' Lectures on Comparative Anatomy ', vols. 1 et 2avec atlas. Londres, 1814.

(8) Macallum, A. B.—" The Alimentary Canal and Pancreas of Acipenser,Amia, and Lepidosteus ", ' Journ. Anat. and Phys.', 20, 1886.

(9) Monro, H.—' The Structure and Physiology of Fishes, explained andcompared with those of Man and other Animals '. Edinburgh, 1785.

(10) Miiller, J.—" Vergleichende Anatomie der Myxinoiden, Untersuch-ungen iiber die Eingeweide der Fisehe ", ' Abh. d. K. Akad. d.Wissensch. zu Berlin ', 1843.

NO. 290 T

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(11) Oppel, A.—' Lehrbuch der vergleichenden mikroskopischen Anatomieder Wirbelthiere ', 1 Theil: der Magen, Iena, 1896 ; 2 Theil:Schlund und Darm, Iena, 1897.

(12) Pilliet, A.—" Note sur l'estomac des Pleuronectes ", ' C. R. de laSoc. de Biologie ', 9e ser., torn, x, 1893.

(13) " Recherches histologiques sur l'estomac des Poissons osseux(Pleuronectes) ", ' Journ. d'Anat. et de Physiol.', torn, xxx, 1894.

(14) Rathke, H.—' Ueber den Darmkanal der Fische ', Halle, 1824.(15) ' Bemerkungen iiber den Bau des Amphioxus lanceolatus'.

Konigsberg, 1841.(16) Rollett, A.—" Bemerkungen zur Kenntniss der Labdriisen und der

Magenschleimhaut", ' Untersuchungen aus d. Instit. fur Physiol.und Histol. in Graz ', 1871.

(17) Stirling, W.—" On the Chemistry and Histology of the DigestiveOrgans of Fishes ", ' 2nd Annual Report of the Fishery Board forScotland1, 1884.

(18) Sullivan, M. X.—" The Physiology of the Digestive Tract of Elasmo-branchs ", ' Bull, of the Bureau of Fisheries ', vol. xxvii, 1907.

(19) Valatour, M.—" Recherches sur les glandes gastriques et les tuniquesmusculaires du tube digestif dans les Poissons osseux et les Batra-ciens", 'Ann. des Sc. nat.', 4e ser., torn, xvi, 1861.

(20) Vonk, H. J., jun.—" Le role des glandes de Lieberkiihn en rapport avecla digestion ohez les Poissons ", ' Arch, neerl. de physiol.', 12, 1927.

(21) Yung, E.—"Recherches sur la Digestion des Poissons", 'Arch, deZool. Exper. et Gen.', 3e ser., torn, vii, 1899.

The Histology of the Alimentary Tract of the Plaice (Pleuronectes

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