Note on the Ciliated Pit of Ascidians and its Relation to the Nerve-ganglion and so-called Hypophysial Gland; and an Account of the Anatomy of Cynthia rustica (?)

Amaræcium Embryo.—The embryo remains in the atrial cavity of the parent until it has attained to the fully-developed tadpole stage.

The nervous system then consists of four parts:

1. An anterior dorsal part (fig. 3 (1)), exactly resembling in structure the ganglion of the adult.

2. A mass (fig. 3 (2)) lying ventral and posterior to (1), composed of very large ganglion cells with very distinct nuclei and nerve-fibres.

3. A nerve-cord (fig. 3 (3)) passing off from (2) into the tail.

4. A hollow sense-vesicle lying to one side of (2), and consisting of a vesicle with thin anterior and thick posterior walls. The unpaired eye is embedded in the wall at its antero-dorsal angle, and the otolith is situated on its floor and projects upwards into its cavity. This is the only part of the nervous system which is hollow at this time.

The ciliated pit opens into the buccal cavity and thence passes back, lying ventral to the anterior part of the nervous system, penetrates the junction between (1) and (2), and continues its course on the dorsal side of (2), ending blindly (E.) not far from the atrial pore (A.P.). At two points it opens into the solid nervous substance: first (5,), at about the middle point of the ventral surface of (1); secondly (R.), on the dorsal surface of (2).

Maurice and Schulgin¹ failed to find the ciliated pit in the embryo of Amaræcium, and state that there is no connection between the buccal cavity and the nervous system. This connection was quite clear in all my sections in which the stomodæum and nervous system were definitely established.

Kowalevsky² states that in Phallusia mammillata the mouth communicates with the hollow anterior end (viz. sense-vesicle) of the nervous system by a pore, which eventually gives rise to the ciliated pit. It is possible that he may have overlooked the existence of the ciliated pit in the embryo, as in optical sections, with which he worked almost entirely, it might appear as a simple pore.

Seeliger¹ observed the pit in the embryo Clavellina, though he found no connection between it and the nervous system.

Salensky² describes the ciliated pit in the embryo of Sal pa democratica as a short tube forming a communication between the pharynx and the anterior end of the nervous system, which is at first hollow, but afterwards becomes solid.

Kupffer³ found no communication between the mouth and nervous system in the embryo of Ascidia mentula.

Van Beneden and Julin⁴ describe the ciliated pit in the embryo of Clavellina rosaceus, and also state that on its ventral side it communicates with a mass of tissue lying ventral to it which gives rise to the gland. From their figures I believe this mass of tissue to be homologous with what I have described above as the posterior ventral part of the nervous system in Amaræcium. In the latter I assume that this structure is a part of the nervous system on account of its histological features, and also from the fact that it connects the dorsal part of the nervous system with the nerve-cord of the tail.

I have not worked out the development from the oldest unhatched embryo into the adult form owing to lack of material, so that the views here put forward must be merely provisional.

There can be little doubt that the part of the embryonic nervous system, which is found persisting in the adult, is the anterior dorsal part, this being rendered probable both by the histological resemblance of the two structures, and also by the fact that their position with regard to the ciliated pit is the same in both cases.

The adult condition most nearly resembling that of the embryo Amaræcium is found in Clavellina. Here the ciliated pit retains its connection with the nervous system, communicating with the ganglion in precisely the same way as it communicates with the anterior part of the nervous system in the embryo Amaræcium. In both cases it then becomes narrower, passes on and ends blindly—the difference being that in Clavellina it communicates on its ventral side with the gland, in the Amaræcium embryo with the posterior ventral portion of the nervous system. The relation of the ciliated pit to the gland in Clavellina is identical with that of the ciliated pit to the posterior ventral part of the nervous system in the Amaræcium embryo.

In the adult Amaræcium the position occupied in the embryo by the posterior part of the brain and in Clavellina by the gland is filled with a mass of degenerated tissue, and the communication between the ciliated pit and the nervous system has been lost. In Ascidia and Ciona the degenerated tissue is replaced by a mass of somewhat complicated glandular tissue lying under the ventral wall of the ciliated pit and communicating with it.

In Phallusia mammillata the condition is still more complicated, the gland communicating with the peribranchial cavity by a number of secondary funnels, its opening into the mouth being comparatively small.

Judging from the fact that in the embryo Amaræcium the ciliated pit is connected exclusively with the brain, it seems probable that its original function was the aeration of the brain; this mode of aeration being similar to that found in Nemertines. It is doubtful whether it originally opened to the exterior, and was subsequently involved in the stomodæum, or whether its opening into the mouth is primitive. In Amaræcium, at any rate, it is almost certainly epiblastic in origin, as it is derived from the epithelium of the stomodæum and not from the pharynx, as has been stated by Seeliger¹ for Clavellina.

Since, as mentioned above, it has in the late embryo no connection with any other structure than the brain, any other connection which exists in the adult is probably secondary.

In the adult no trace of the posterior part of the brain is found, but occupying its place in Amaræcium is a mass of degenerated tissue, which is connected with the exterior by means of the ciliated pit.

In Ascidia and Ciona, and apparently most other simple Ascidians (cf. Julin, loc. cit.), the function of the ciliated pit is to act as a duct for the so-called hypophysial gland (Julin) which lies in the position occupied in the Amaræcium embryo by the posterior part of the brain.

In Clavellina the ciliated pit has a twofold function.

1. It communicates with the brain, and probably aerates it.

2. Its posterior part acts as a reservoir to carry off the secretion of the gland.

There is thus a gradual transition from one function to another in the different types; the primitive condition, as an organ for the aeration of the brain, is found in the Amaræcium embryo, and is retained in Clavellina, while in the latter the secondary function, viz. that of an excretory duct, is also acquired.

In most adult forms (e.g. Amaræcium, Ascidia, and Ciona) the primitive function is lost, the secondary one only being retained.

The gland is possibly an altogether secondary structure, being developed to supply the need of an excretory organ in the anterior part of the body. It reaches its highest degree of complication in Phallusia mammillata.

If the excretory function of the ciliated pit be merely secondary no homology can exist between it and the proboscis pore of Balanoglossus,¹ or the external opening of the left anterior pouch from the fore-gut, described by Hatschek² in Amphioxus.

It is more probably homologous with the hypophysis of Vertebrates, the original functions of which may have been the aeration of the brain. When a complete blood-supply to the head was effected aeration by this means would no longer be required, and since a definite and complete excretory system had been at the same time developed, there would no longer be any necessity for an excretory organ in this position. Thus the hypophysis at the present time may represent merely a rudimentary condition of the gland and ciliated pit in Ascidians, having almost atrophied, and quite lost its function as a consequence of the development of the ordinary Vertebrate excretory system.

It is possible that the pineal gland of Vertebrates may represent the dorsal continuation of the ciliated pit in the embryo Amaræcium (fig. 3, E).

Whilst investigating the condition of the ciliated pit in various genera of Ascidians, I observed several features in Cynthia, which, as far as I know, have not hitherto been described. I have therfore thought it worth while to publish a short account of the general anatomy and histology of the species I have studied, which I believe to be Cynthia rustica.

N.B.—Some of the generic characters, in which the Cynthiidæ differ from the Molgulidæ, have been pointed out by Lacaze-Duthiers;¹ and Herdman² gives a short account of some forms found by the Challenger.

The individuals are small, varying in length from half to little more than an eighth of an inch, and each is almost as broad as it is long. It is wide and flattened at its base, by which it is attached to the rock, there being no stalk or peduncle. A great number of individuals live upon the same piece of rock, and are very closely applied to one another by their sides, so as to form a compact mass; but any one can be easily separated from the rest. The lateral pressure to which they are thus subjected causes the individuals to vary much in shape.

In colour they are a bright brownish red. They are quite opaque, so that it is not possible to make out any of the internal structure without removing the test.

The oral and atrial apertures are four-lobed.

The test, which gives the red colour to the animal, is fairly thin, and in life is so closely applied to the body wall that it is difficult to remove it without tearing the epidermis. After preservation it can be removed with comparative ease, although it still adheres to the body wall. Except at its base, where fragments of the rock on which it lived generally remain attached to it, the test is free from sand, calcareous spicules, or other foreign matter.

As seen in sections it is composed of a homogeneous matrix with a few scattered cells. It also possesses a complicated system of vessels, which are lined by short columnar cells. In picrocarmine-glycerine preparations of. the test these vessels are clearly seen, as well as numerous pigment cells, which are of two kinds, one appearing dark brown or black, and the other a bright orange.

Beneath the test and closely applied to it is a layer of columnar cells (figs. 12 and 15, Ep.) with very definite nuclei. Their internal ends are rounded and they do not rest upon any basement membrane.

Beneath the epidermis is a thin layer of circular muscle-fibres (fig. 15, c. m.), then a thin layer of longitudinal fibres (fig. 15, I. m.). Within the latter is a very thin layer of circular fibres (fig. 15, c. m.), and then a thick layer of longitudinal and oblique fibres (fig. 15, I. m.), which are arranged in large irregular masses, not definitely marked off from one another, or from the surrounding tissue (fig, 15). All the muscles are unstriated.

A mass of connective tissue, constituting a meshwork of fibres, fills up the spaces between the muscles and extends inwards as far as the lining of the atrial cavity (figs. 12 and 15 Ps.). Nuclei are sometimes visible (fig. 15, c. t. c.) at the points of junction of the fibres; these apparently are the nuclei of the connective-tissue cells, the cell protoplasm being so drawn out and branching as not to be apparent round them.

Among the meshes of the connective tissue and lying freely in it are three kinds of cells:

1. Cells filled with black pigment (fig. 15, p. c.). A nucleus is present in each.

2. Large, coarsely granular cells (fig. 15, g. c.), in which I have not been able to discover nuclei.

3. Cells which stain faintly (fig. 15, b. c.), and have large, deeply-staining nuclei. The cell protoplasm is very finely granular.

Thin laminae of this connective tissue (fig. 12, Ps.₂) pass across the atrial cavity into masses of similar tissue (fig. 12, Ps.₁]), which surround the alimentary canal, thus acting as mesenteries.

Large, irregularly-shaped processes of this connective tissue (figs. 12 and 15, Ps. p.) project into the atrial cavity along its outer wall, being only separated from it by the epithelial lining of the atrial cavity (figs. 12 and 15, A. Ep.) which covers them. Figure 12 is a diagrammatic transverse section through Cynthia, and represents the relation of these processes to the atrial cavity. They appear very conspicuously when the atrial cavity is opened, especially in the fresh animal. They are then seen as very large, opaque, white bodies, which fill up a considerable portion of the cavity. I am not aware that any analogous structure has hitherto been described in any Ascidian.

The connective tissue extends from the body wall inwards to the atrial epithelium (fig. 12, Ps.), and also surrounds the alimentary canal. There is therefore no body cavity, its place being occupied by what appears to be a system of sinuses, the third kind of cell described above being the blood-corpuscles. For convenience of description I shall hereafter speak of the sinuses as the pseudocœle; but I do not wish to imply that it is necessarily homologous with the so-called pseudocœle of some Invertebrates. There are no definite blood-vessels, the heart opening out at both ends into the sinuses. Strands also pass across the atrial cavity to the pharynx, the spaces in the branchial bars being continuations of the sinuses, plentifully supplied with blood-corpuscles (figs. 5 and 7, br. b.).

It seems probable that the function of the processes projecting into the atrial cavity is to expose a greater surface of the sinus to the influence of the oxygen contained in it.

The atrial cavity is very capacious, extending beyond the posterior end of the alimentary canal. For the greater part of its extent it is divided completely into two halves by a partition, which passes along from the outer wall of the atrial cavity to the walls of the pharynx. This partition starts just behind the buccal cavity, follows the line of the endostyle, and curves round the posterior end of the pharynx on to its dorsal surface, where it passes along the line of the dorsal lamina, and stops just behind the atrial pore. The whole of the alimentary canal behind the pharynx is situated on the left side of the partition (fig. 12, St. and Int.). Thus the alimentary canal, embedded in its own portion of the “pseudocœle,” is completely surrounded by the atrial cavity except at those points where strands pass off connecting its “pseudocœle” with the “pseudocœle “in the body wall outside the atrial cavity, and forming the mesenteries described above.

The atrial cavity is lined throughout by a layer of flattish cells, lying upon a basement membrane (figs. 12 and 15, A. Ep.). The cells are rounded at their free ends, and have large nuclei. This layer, of course, extends over the portions of the pseudocœle surrounding the alimentary canal, and over the mesenteries.

The atrial pore is placed not far from the mouth on a short papilla.

The mouth leads into the buccal cavity, which passes into the large pharynx (fig. 4, Ph.). The endostyle extends round the posterior end of the pharynx, ceasing at the point where the oesophagus is given off. The oesophagus (fig. 4, Oes.) is short, and soon opens into the large sack-like stomach (fig. 4, St.), which, on its external surface, is marked by deep longitudinal ridges. The stomach lies on the left side of the pharynx, its long axis being at right angles to that of the latter. It passes into the intestine (fig. 4, Int.), which almost at once bends upon itself and runs back across the pharynx, almost parallel with the stomach. On reaching the level of the dorsal side of the pharynx it turns forwards almost at a right angle, and runs straight to the anus (fig. 4, A.), which is situated at a short distance from the atrial pore, at the point where the partition dividing the atrial cavity ceases.

Shortly after leaving the stomach the intestine appears to be dilated for a short distance (fig. 4, Int. L.); this appearance is due to its being surrounded here by a layer of liver-cells.

The buccal cavity is lined just within the mouth by a portion of the test which grows into it. This cellulose lining acquires a covering of thin epithelium, and forms a circle of short, blunt, unbranched tentacles. Further down, the cavity is lined by high ciliated columnar cells, which are thrown up into papillæ. At the junction of the buccal cavity with the pharynx there is a circle of cirri, which are unbranched, and in transverse action are four-lobed (fig. 10), one lobe being much larger than the other three. Two of the small lobes bear tufts of cilia.

Outside the epithelial lining of the buccal cavity there is a layer of connective tissue, and around this a thick layer of longitudinal and circular fibres.

The ciliated pit is simple, having the conditions found in Ascidia and Ciona. Its opening into the mouth is crescentshaped, and it passes back thence, as a simple tube lined by high columnar ciliated cells, below the ganglion, at about the middle of which it opens out into the hypophysial gland, which lies immediately ventral to the ganglion. It has no communication with the latter.

The Pharynx.—The endostyle starts from the point where the buccal cavity joins the pharynx, and passes round its posterior end, ceasing where the œsophagus passes off (fig. 4, End.). It is very similar to the endostyle of other Ascidians, bearing a tuft of very long cilia at its base and shorter ones at the sides of the groove (fig. 9, c. c.). Between the masses of cells, which bear the cilia, are situated groups of much higher cells (fig. 9), while on each side, between the most external masses of ciliated cells and the ciliated epithelium of the pharynx, there is a row of cells which appear to secrete mucous (fig. 9, m. c.), as they appear when seen in transverse section to be throwing out irregular processes towards the cavity. At the anterior and posterior end of the endostyle its open edges are fused, so as to form a tube (fig. 8). This is an interesting point, as tending to confirm the theory of its homology with the thyroid body of the higher Vertebrata.

A thin lamina (the “dorsal lamina covered by low columnar ciliated cells (fig. 5, Z>. L.), projects from the median dorsal line of the pharynx a long way into it; a rod of skeletal tissue (fig. 5, sk. b.) is present near its base, and extends throughout its whole length.

The gill-slits are very numerous. The bars between them are composed of connective tissue, which is a part of the pseudocœle, and contains a great number of blood-corpuscles (figs. 5 and 7, br. b.).

The skeletal system is somewhat complicated. The slits (fig. 7, br. s.) are elongated longitudinally, and arranged in transverse rows. The rows are separated from one another by thick skeletal rods, and divided into sets of five by similar rods (fig. 7, sk. b.), which meet the former at right angles. In addition to this main skeletal system, there is a system of finer rods (fig. 7, s. sk. b.) which accompany the larger ones, and are connected together by longitudinal rods passing between every two gill-slits, and a transverse rod lying across the centre of each row of slits. This secondary system of rods lies on the internal face of the pharynx.

The bars themselves in transverse section (fig. 5, br. b.) are seen to be covered on the surfaces turned towards the slits by columnar cells provided with cilia, and on their inner and outer walls by flat non-ciliated cells. Their internal cavity, as already stated, contains a blood sinus.

The (Esophagus (fig. 4, Πs.) is very short and simple, being-lined with short, columnar, ciliated cells.

The Stomach.—The walls of the stomach are thrown up into deep glands, which run in a longitudinal direction along its whole length.

The mouths of the glands are lined by high columnar ciliated cells (fig. 6, c. c. s.), very closely packed together, with a smallish nucleus placed at about the centre of each cell. The portion of the cells towards the lumen stains very deeply.

These cells are separated from those forming the deeper part of the gland by a slight constriction (fig. 6, c.).

The latter (fig. 6, s. c. s.) are somewhat higher than the former, and are not ciliated. The nuclei are placed quite near the bases of the cells. They appear to be secretory cells, as their protoplasm is rich in granules, and at their free ends many of them are prolonged into blunt processes (fig. 6), which project into the lumen, and only stain very faintly. In most of the cells these processes are not visible, but since they otherwise resemble those that have them, the difference would seem to be due to their being in a different stage of secretory activity. In such cells the end abutting on the lumen stains more deeply than the rest of the cell, and the two regions are separated in preserved specimens by a row of very deeply-staining dots. Among the cells a few leucocytes are scattered (fig. 6, I.). The glands are separated from one another by a portion of the pseudocœle.

The intestine is lined throughout by rather short, ciliated columnar cells (figs. 11, Ep. Int.).

When the intestine is viewed as a whole it is seen to be enlarged for a short distance (fig. 4, Int. L.) soon after leaving the stomach. This appearance is due to its being surrounded by the so-called liver-cells. These are large and oval, are closely applied to the wall of the intestine, and form a compact mass round it. In the fresh state they are bright orange in colour. Each cell is pear-shaped with its thin end in contact with the epithelium of the intestine (fig. 11, L.), and is surrounded by a somewhat thick fibrous coat (f. I.), which is thickest at the broad end of the cell. In preserved specimens the cells are filled with a coarsely granular substance (fig. 11, ff. I.), which is mostly aggregated at the narrow ends, and contains highly refractive concretions.

I could not find any connection of these cells, either with one another or with the intestine, but it seems possible that as they lie so closely applied to the wall of the intestine, their contents may pass into its lumen between the epithelial cells, the passages being so small as to have escaped my notice. I am unable to offer any suggestion as to their probable function.

The heart is a long, thin-walled vessel, lying in a pericardium, which is considerably larger than itself.

It curves round the posterior end of the body, following the line of the partition between the two halves of the atrial cavity. For the greater part of its length it is situated in the pseudocœle external to the atrial cavity, but the anterior end of it lies in the partition, i. e. immediately ventral to the endostyle. It extends backwards nearly as far as the oesophagus.

At both ends it opens out into the pseudocœle, anteriorly by a long narrow slit in its dorsal wall, and posteriorly by a terminal pore, so that there is apparently no closed system of blood-vessels, but the circulation is carried on only through the blood-sinuses which surround all the organs and fill all the spaces usually occupied by the body cavity.

The pericardium is entirely closed.

The same condition of a heart situated in a closed pericardium, and itself opening at both ends, is said by Seeliger¹ to exist in Clavellina. The gradual shutting off of the heart from the body cavity in the development of the embryo, as he describes it, explains how the adult condition may have been brought about in the case of Cynthia.

The generative organs are unpaired, and lie near the posterior end of the body in the pseudocœle, outside the right half of the atrial cavity (fig. 12, 0. and t’s.).

They are arranged in two long masses on the outer and inner sides of a cavity (fig. 14, G. D.), which is lined by flat cells, and opens posteriorly into the atrial cavity. The mass on the outer side consists of testes (figs. 12 and 14, is.), that on the inner of ova (figs. 12 and 14. O.).

The testes (fig. 13) are large oval sacs filled with spermatozoa (fig. 13, sp.); each sac is lined by a thin membrane, outside which are bundles of muscles (M. ts.), and, especially at its outer end, large collections of pigment-cells. At its inner end, i. e. towards the cavity, it is drawn out into a short duct (figs. 13a, 14, D. ts) lined by low columnar cells, which terminates close to the wall of the cavity, but does not appear to open into it. It is possible that when the spermatozoa become ripe they pass down the duct into the cavity by an opening, which is temporarily established into it, the communication being closed at other times.

The ovary (fig. 14, 0.) lies on the opposite side of the cavity. It has no limiting membrane, the ova lying freely in the pseudocœle. There is no oviduct, but the ova, when ripe, probably break through the wall into the cavity, which thus serves as the common generative duct, conveying both male and female products into the atrial cavity.

Each ovum is surrounded by a single layer of cells, which either are follicle cells or give rise to the test.

The nervous system has the form generally found in adult Ascidians. It consists of a large ganglion lying between the mouth and atrial pore, and is composed of fibres, which are surrounded by a peripheral layer of ganglion cells. Anteriorly it sends off two nerve trunks, which pass, one on each side of the mouth; posteriorly it also sends off two trunks, which very soon divide again into several small branches, whose course I could not follow for any distance.

The hypophysial gland is a compact mass of tissue, lying immediately ventral to the ganglion. It consists of a great number of fine glandular tubes, which towards the dorsal side of the gland unite, forming larger ducts, which open into the canal of the ciliated pit.