The anatomy of the gut in Crania anomala Müller was described by Joubin (1886) and Blochmann (1892), both of whom also made some observations on its histology. The present paper gives an account of the structure of the gut and the results of some histochemical tests.
MATERIAL AND METHODS
Specimens of C. anomala were dredged off Little Cumbrae, Scotland. The adults were removed from the stones to which they were attached, and fixed at once at the Marine Station, Millport. Histochemical tests were carried out in the Cytological Laboratory, University of Oxford. Details of these tests are given in the appendix (table 1).
Anatomy of the gut
The gut in C. anomala is longer than the visceral cavity. The mouth is a horizontal slit situated in the sagittal plane where the two brachial grooves meet, as Joubin (1886) and Blochmann (1892) noted. The pharynx (fig. 1, A), into which the mouth opens, is a dorsally convex curved cylinder embedded in the bases of the brachia. Sinuses communicating with the visceral cavity abound in the pharyngeal connective-tissue envelope. The gut leaves the bases of the brachia as a short straight oesophagus (fig. 1, A) of uniform diameter. This lies medially in the anterior part of the visceral cavity and is surrounded by a thin sheath of compact connective tissue. The stomach (fig. 1, A) increases in diameter from the oesophageal end, near which it receives antero-dorsally the ducts of the two digestive diverticula. The stomach reaches its greatest diameter just in front of a marked constriction.
Posteriorly it narrows and eventually becomes a tube of uniform diameter. This narrow tube curves ventrally towards the left. A slight constriction near the end of the curved region indicates the position of a sphincter (fig. x, A-D), which marks the beginning of the intestine.
The intestine is V-shaped with its vertex directed anteriorly (fig. 1, B). From the sphincter it increases in diameter only to taper again near the anus. When distended, it assumes grotesque shapes as a result of unequal dilation of its various regions, and its anterior portion then tends to be directed transversely to the longitudinal axis of the animal (fig. 1, D). The anus (fig. 1, B-D) opens into the mantle cavity posteriorly, as Joubin (1886) first noted. The last part of the intestine lies in a conical posterior extension of the visceral cavity. This conical extension lies to the right of, and behind, the place of attachment of the dorsal and ventral mesenteries to the intestine. Hence the last part of the intestine lies in the right half of the visceral cavity, as in other inarticulates.
There are two digestive diverticula separated by a median dorsal mesentery. The right one arises from the antero-dorsal surface of the anterior chamber of the stomach to the right of the dorsal mesentery by a wide duct, which immediately branches into two to serve the two constituent lobes of the diverticulum—an anterior and a posterior lobe. The anterior lobe (Joubin’s superior and middle lobes) usually consists of 7 lobules of different sizes. The posterior lobe (Joubin’s inferior lobe) comprises 6 lobules. The left diverticulum arises from a corresponding position to the left of the dorsal mesentery usually by two ducts which lie in an antero-posterior direction (fig. 1, A). The anterior duct serves the anterior lobe (Joubin’s superior and middle lobes) of 9 lobules; the other serves the posterior lobe (Joubin’s inferior lobe) of 8 lobules. Each lobule is made up of a number of small branched ductules and acini. The lobulation is not regular, as in many specimens there are some isolated branched ductules and acini arising near the bases of the big ducts. Moreover, lobulation of the more ventral part of each lobe is variable.
A dorsal mesentery (fig. 1, A) extends from the mid-dorsal surface of the gut to the dorsal body-wall. A ventral mesentery (fig. 1, A) descends mid-ventrally from the pharynx, oesophagus, and stomach to the bases of the brachia and the ventral body-wall, but along the curved part of the intestine it extends antero-ventrally to the ventral body-wall. The dorsal and ventral mesenteries divide the visceral cavity into two compartments and terminate some distance from the anus. Since the line of attachment of the ventral mesentery along the anterior part of the intestine is directed laterally towards the right, the greater part of the originally left lateral side of the intestine faces ventrally instead. This presumably is caused by asymmetrical growth of the anterior part of the intestine during development, which produces a clockwise rotation through about 90° when viewed from the anterior end.
A low longitudinal ridge of connective tissue, which may be called the longitudinal gastro-parietal band (Blochmann’s ileo-parietal band), extends along each side of the oesophagus and stomach. Near the constriction between the two chambers of the stomach the band, continuing in the same direction, leaves the surface of the stomach. It crosses and overlies a narrow transverse band of connective tissue, which may be called the transverse gastro-parietal band (gastro-parietal band of Blochmann (1900)), that extends laterally from the mid-dorsal region of the stomach just behind the constriction. On each side the longitudinal band fuses with the transverse gastro-parietal. About 1 mm from this point of fusion the former expands posteriorly into a large triangular piece of transparent membrane, whose lateral edge becomes attached to the lateral body-wall and whose lower surface suspends the gonad. The other band continues ventrally forward on each side to support the nephridium.
Histology of the gut
The gut is lined internally by a simple columnar epithelium resting on a basement membrane, which forms the innermost layer of the connective-tissue stroma. In the free part of the gut this is invested with squamous mesothelium, in many regions of which cilia have been observed, as Blochmann (1892) previously noted. Joubin (1886) mentioned ‘la gaine de cartilage’, which corresponds to either the basement membrane or the connective-tissue stroma of this paper.
The inner epithelium of the gut shows circular and longitudinal folds in the pharynx, but only longitudinal ones in the oesophagus. Between this and the stomach is a circular fold where the nuclei on the oesophageal side are slender and long as in the oesophageal epithelium, while those on the stomach side are shorter and stouter as in the stomach epithelium. Two parallel grooves run transversely across the dorsal and lateral walls of the anterior chamber of the stomach, approximately equidistant from the openings of the digestive diverticula and the gastric constriction. The epithelium that intervenes between these grooves has the form of a fold, which reminds one of a similar but shorter curved fold that runs along the right wall of the anterior chamber of the stomach in Lingula (Chuang, 1959, fig. 8, a). A ciliated epithelial groove runs longitudinally along the floor of the anterior chamber of the stomach and continues postero-dorsally from the floor to the roof of the posterior chamber by way of the right lateral wall (Chuang, 1959). The intestinal epithelium bears some circular folds, especially when the intestine is not fully distended.
The columnar epithelium is ciliated everywhere except in the acini of the digestive diverticula, and consists of ordinary epithelial cells interspersed with goblet cells and occasional wandering phagocytes. In the pharynx and the oesophagus the ordinary epithelial cells are tall and slender, with elongated nuclei (fig. 2, A). Shorter cells with shorter, broader nuclei line the stomach and the intestine. In the stomach these cells have a distinct border formed of the prominent roots of strong cilia. This border is widest and most prominent in the funnel-shaped region of the posterior chamber of the stomach, becoming narrower and less conspicuous both anteriorly and posteriorly. Short columnar cells with subspherical nuclei occur in the digestive diverticula (fig. 2, B, c), but whereas the distal ends of the cells in the ducts are flat, those in the acini bulge out in varying degrees to form an uneven surface. Blobs of cytoplasm break away from the domes of these acinar cells to appear in the lumen, as in Lingula unguis (Chuang, 1959).
The goblet cells are especially abundant in the stomach, intestine, and floor of the pharynx (fig. 2, D), but comparatively scarce in the digestive diverticula (fig. 2, E) and the oesophagus. They usually lie with their distended part between the bases of neighbouring cells. Those with few small globules tend to have large subspherical nuclei, while those with numerous large globules have small flattened nuclei.
In the connective-tissue stroma are found a basement membrane on the inner epithelial side, a network of connective-tissue fibres and cells in the middle, and a network of separate muscle strands externally. The connective-tissue fibres are fine, single, branching fibres. The muscular network consists of an inner layer of broad strands arranged circularly and an outer layer of narrow strands arranged longitudinally along the gut, with the former pre-dominating. This muscular network is especially well developed in the pharynx, where the circular strands are most conspicuous. The digestive diverticula, though less muscular than the pharynx, are as muscular as the stomach. Presumably owing to dilation, the intestine appears the least muscular part of the alimentary canal, especially in its anterior region.
Of the cytoplasmic inclusions in the gut-wall only the pigment granules and the subspherical globules are easily recognizable under the light microscope. These pigment granules occur in the epithelium, connective-tissue cells, mesothelium, and lumen of both the digestive diverticula and the stomach. They are irregularly shaped inclusions in various shades of yellow, about 2 μ in diameter. The subspherical globules vary in diameter from less than 1 μ to about 12 μ and can conveniently be divided into two groups. One group of globules, henceforth called lipochondria, varies from less than 1 μ to about 3 μ in diameter. They occur in the ordinary epithelial cells throughout the gut, in the detached blobs of the acinar cells in the digestive diverticula, and in the connective-tissue cells and mesothelium. The other group comprises the globules confined to the goblet cells (fig. 2, D, E); these measure from less than 1 μ to 12 μ in diameter. Food vacuoles have been observed to occur singly in the distal part of the acinar epithelium.
Histochemistry of the gut
Amino-acids and proteins. The nucleus and cytoplasm of all the cells of the gut were weakly positive to the Hg/nitrite test for phenols (Baker, 1956), and the modified Sakaguchi test for arginine (Baker, 1947). They therefore contain some tyrosine and arginine. The tests also indicate the presence of tyrosine and arginine in the ground substance, fibres and basement membrane of the connective tissue in the gut.
The globules in the goblet cells gave a positive reaction to the acid haematein test after pyridine extraction (fig. 2, D, E), the Hg/nitrite test for phenols, and the modified Sakaguchi test for arginine. They also gave a positive reaction to PAS, which was not affected by amylase. They showed no metachromasy with toluidine blue. Hence the protein in these globules should be regarded as a muco- or glycoprotein (Pearse, 1953).
Fine, irregular granules in the nuclei of cells (fig. 2, A, B) were positive to the Feulgen test for DNA (Feulgen and Rossenbeck, 1924). These DNA granules were sparse in the nuclei of the intestinal epithelium, denser but variable in the epithelial nuclei of other regions of the gut, and densest in the nuclei of the mesothelial and connective-tissue cells.
Similar granules positive to the Feulgen test were found in the cytoplasm of the epithelial cells in the digestive diverticula. These extranuclear DNA granules had a wider range of sizes than the intranuclear ones. Many of them occurred in the neighbourhood of nuclei deficient in DNA (fig. 2, B), although a few were found in the distal part of the cytoplasm. This therefore provides evidence for the belief that these cytoplasmic DNA granules originated from the nuclei and that they migrated towards the lumen.
The weakly positive reaction to PAS of some extremely minute granules in the distal part of the gut epithelium from the pharynx to the stomach, including the digestive diverticula, was abolished by previous incubation of the slide with saliva as control. These granules therefore contain a carbohydrate soluble by amylase, presumably glycogen.
The connective tissue of the gut gave a positive reaction to both the Hg/nitrite test for phenols and the modified Sakaguchi test for arginine. The basement membrane and the fibres most probably contain an absorbed acid mucopolysaccharide, because of their strong γ-metachromasy with toluidine blue and their amylase-fast positive reaction to PAS. After treatment with chromic acid or sulphuric acid, the γ-metachromasy of the basement membrane and the connective-tissue ground substance was increased; these structures therefore presumably also contain some neutral mucopolysaccharide.
A negative reaction to the standard tests for lipids was given by the ground cytoplasm of the gut cells. The lipochondria of the epithelial cells were positive to Sudan IV, Sudan black, and the acid haematein test (Baker, 1946). These lipochondria contain a phospholipid, since their positive reaction to the acid haematein test is abolished by previous pyridine extraction.
The gut of C. anomala does not differ much from the gut of other recent inarticulate brachiopods. In this paper the different regions of the gut are named by homology with those in Lingula (Chuang, 1959). The relationship to the terms used by Joubin (1886) and Blochmann (1892) is summarized in table 2 of the appendix.
Although the anus opens posteriorly in the median line—a character that Blochmann (1892) regarded as primitive—a short posterior part of the intestine, situated to the right of both the dorsal and the ventral mesenteries, actually lies in the right half of the visceral cavity, as in other recent inarticulates. The stomach, unlike that in Lingula, does not reach the posterior end of the visceral cavity, nor does it show as much morphological difference between its two chambers as that of Lingula. The greater part of the posterior chamber of the stomach and the short, distended intestine are of comparatively larger diameter than the corresponding regions in Lingula. Presumably it was necessary for C. anomala to make good the reduction in capacity through a decrease in length by an increase in diameter of some regions of the gut. Presumably all these and the characteristic, posterior position of the anus may be an adaptation necessitated by the greatly reduced visceral cavity.
The glycogen reported in the epithelium of the gut from the pharynx to the stomach, including the digestive diverticula, is a reserve food store, synthesized from food carbohydrate, as Chuang (1959) has reported the presence of an amylase acting on carbohydrate in Lingula. The globules of muco- or glycoprotein produced by the goblet cells are extruded into the lumen of the gut presumably for the lubrication of the epithelium and the entanglement of food particles.
The inarticulate gut is a difficult tissue for the demonstration of muscle strands, as Chuang (1959) managed to demonstrate the presence of the circular and longitudinal strands only after trying several staining methods. Thanks to Baker’s acid haematein test following pyridine extraction (1946), which stains the proteins of the muscle strands but leaves unstained the basement membrane, fibres, and ground substance of the connective-tissue sheath, it is possible to demonstrate them in the digestive diverticula of C. anomala (fig. 2, E). Blochmann (1892) remarked, with reference to these fibres, ‘Bei den Leberlöppchen selbst konnte ich keine Muskelfasern mehr nachweisen.’ The entire gut-wall has been found to possess both circular and longitudinal muscle strands. The circular strands are most prominent in the relaxed pharynx and oesophagus, where the broad circular strands form distinct sheets, whereas the thin longitudinal strands remain separated from one another. Blochmann’s failure to find one set of muscle strands or the other was presumably due to the different degrees of contraction of the various regions of the gut.
There is evidence of intracellular digestion in the digestive diverticula, since numerous food vacuoles were observed in the distal part of the acinar epithelium in C. anomala. The pulsation cycles, which are responsible for the transport of suspended particles between the stomach and the digestive diverticula in Lingula (Chuang, 1959), could very well occur in Crania, since the wall of the digestive diverticula in Crania appears more muscular than that in Lingula.
The cytoplasmic inclusions of the gut epithelium include pigment granules, glycogen granules, lipochondria, and goblet-cell globules. The lipochondria contain a phospholipid. The goblet-cell globules contain a muco- or glycoprotein, and are extruded into the lumen of the gut presumably for lubrication and for the entanglement of food particles. Extranuclear DNA, presumably originating from the nucleus, occurs in the cytoplasm of the ordinary epithelial cells in the digestive diverticula.
The gut of Crania anomala has been studied morphologically and histochemically. It is attached to the body-wall by dorsal and ventral mesenteries with the exception of the posterior part of the intestine, which lies free in the right half of the visceral cavity. The gut-wall consists of an inner columnar epithelium, a connective-tissue stroma, and an investing squamous mesothelium. The columnar epithelium comprises ordinary epithelial cells, some goblet cells, and occasional phagocytes.
I wish to thank Dr. J. R. Baker, F.R.S., under whose supervision this work was carried out, for the help I have received and the laboratory facilities enjoyed. My thanks are due also to Professor Sir A. C. Hardy, F.R.S., for facilities in his department, to Mrs. B. M. Jordan-Luke and Dr. J. T. Y. Chou for many helpful suggestions, and to Dr. A. P. Orr, Deputy Director, and the staff of the Marine Station, Millport, for the supply of the specimens, and especially Mr. E. Latham for fixing some of them. The work was carried out during study leave granted by the University of Malaya.