Stolonic test vessels are described in four members of the ascidian family Didemnidae. The epidermal cells of the terminal swellings of the vessels appear to produce test substance. The stolonic vessels, area of budding and muscular appendix are probably all derived from the posterior end of the ancestral zooid.

This paper deals with the position, structure, and function of the stolonic test vessels in four members of the ascidian family Didemnidae.

These vessels, along with the muscular appendix arising just behind the posterior end of the endostyle, are structures associated, in most other ascidians, with the extreme posterior end of the body. The forward position of the stolonic vessels and the appendix in this family may be regarded as a result of dwarfing of the zooid. The appendix has been thought to represent a much reduced post-abdomen, but this interpretation depends on accepting the Didemnidae as having been derived from the polyclinid stock rather than from the clavelinid stock. Of the three aplousobranch families the Clavelinidae is the most primitive, the Polyclinidae having been derived from this stock, but the evolution of the Didemnidae from the Polyclinidae is uncertain. It is perhaps easier to derive the Didemnidae from the clavelinid stock through forms similar to Distaplia. We cannot therefore refer with confidence to the stolonic vessels and the appendix together as the post-abdomen, but they are certainly structures derived from the extreme posterior part of the body.

Vascular stolons or test vessels are prominent in the test of many ascidians. They are tubular epidermal prolongations of the body-wall and contain some mesenchyme hollowed out to form blood channels. In the Didemnidae the stolonic vessels do not differ essentially from the test vessels of other ascidians, but are modified in ways to be described.

The material was collected from the littoral zone of various parts of the west coast of Scotland. The species studied were Diplosoma listerianum (Milne Edwards), Lissoclinum argyllense Millar, Didemnum maculosum (Milne Edwards), and Trididemnum tenerum (Verrill).

Diplosoma listerianum

The colony of Diplosoma has an upper and a lower layer of test between which the zooids are suspended in a narrow sheath of test substance (fig. 1B). The stolonic vessels take the form of several narrow tubes, each with a dilated end (fig. 1C, D). These tubes, which arise from the body-wall covering the oesophagus and anterior part of the stomach, may be quite short or may penetrate down into the basal layer of common test. They are totally embedded in test matrix.

FIG. 1.

Diplosoma listerianum. A, zooid from a young colony; B, zooid from an old colony; c, zooid showing stolonic vessels, muscular appendix, and bud; D, terminal swelling of stolonic vessel lying within strand of test; E, two cells from the secretory pad of the terminal swelling.

FIG. 1.

Diplosoma listerianum. A, zooid from a young colony; B, zooid from an old colony; c, zooid showing stolonic vessels, muscular appendix, and bud; D, terminal swelling of stolonic vessel lying within strand of test; E, two cells from the secretory pad of the terminal swelling.

Each stolonic vessel consists of a tube of cubical epidermal cells, the diameter of the tube usually being between 6μ and 15μ. Within the epidermal tube there is mesenchyme with blood spaces. The vessel ends in a bulb-like swelling, the distal part of which consists of a pad of tall narrow cells. These cells (fig. 1E) have a narrow tapering basal part. The nucleus lies in the basal third of the cell and is spherical with a large rounded central mass of chromatin. The part of the cell immediately distal to the nucleus colours deeply with basic stains but towards the apex the staining reaction is less intense. One or two indistinct vacuoles are generally visible between the nucleus and the apex of the cell. In the apical part of the cell are several deeply staining rounded bodies, which are probably the precursors of a secretion. This secretion is liberated from the apex of the cell as.a rounded and rather pale droplet. When a piece of test is examined which contains the dilated end of a stolon, a clear area can be seen round the pad of secretory cells. In this area no cells or fibres are present, although in all parts of the surrounding test they are abundant. This clear area is interpreted as a zone of newly secreted test substance into which cells and fibres have not yet penetrated.

Lahille (1890), who saw the terminal swellings of the vessels, suggested that they might produce tunicin, although he neither described their structure nor gave any evidence for his opinion. There seems little doubt, however, that this is their function, and indeed they provide one of the few demonstrations of the secretion of test material by epidermal cells.

The situation of these localized centres of test production has a considerable effect on the form of the colony. In young colonies (fig. 1A) the oral siphon is embedded in the upper layer of the common test and the abdomen reaches the lower layer. As the stolonic vessels grow downwards their ends come to lie below the abdomen so that test matrix is added between the abdomen and the lower layer of test, producing a lengthening column under the zooid. This is one of the factors responsible for the very extensive common cloacal cavities within old colonies. A similar case of the influence of the position of the stolonic vessels on the form of the colony has been noted by Berri1l (1935) in Clavelina, but in that genus immigrant mesenchyme cells are the agents of test production.

In Diplosoma it seems that a series of these stolons grows downwards from the zooid during the life of the colony, each one adding its quota to the common test. This is shown by the presence of a number of terminal bulbs lying at different levels between the zooid and the basal layer of test. There is evidence in Diplosoma and in other genera that one or more new stolons are produced each time a bud is formed. This would account for the serial production of stolonic vessels over a period of time.

Lissoclinum argyllense

Lissoclinum is more closely related to Diplosoma than to Didemnum or Trididemnum. The form of the colony is one of the characters showing the relationship between Lissoclinum and Diplosoma. In Lissoclinum the zooids (fig. 2A) of an old colony are united to the upper layer of test by their oral siphon and to the lower layer by a long narrow strand of test substance. Stolonic vessels extend, as in Diplosoma, downwards below the abdomen of the zooid, and produce test substance which forms the long strand between the abdomen and the base of the colony. In the colonies examined vessels tended to occur in pairs, suggesting that on a series of occasions two vessels were produced simultaneously.

FIG. 2.

Lissoclinum argyllense. A, zooid showing stolonic vessels and buds; B, longitudinal section through abdomen of zooid, showing origin of stolonic vessel and bud; c, cells from the terminal swelling of a stolonic vessel, showing threads of test substance between test and apex of cells.

FIG. 2.

Lissoclinum argyllense. A, zooid showing stolonic vessels and buds; B, longitudinal section through abdomen of zooid, showing origin of stolonic vessel and bud; c, cells from the terminal swelling of a stolonic vessel, showing threads of test substance between test and apex of cells.

The exact point of origin of the stolonic vessels can be clearly seen in sections of Lissoclinum (fig. 2B). A small area of epidermis, situated laterally between the stomach and the lower part of the intestine, is responsible for producing buds and stolonic vessels. Buds arise from the anterior part of this area and stolonic vessels from the posterior part. It is reasonable to suppose that when buds are being formed this area of proliferation also produces new stolonic vessels.

In structure the vessels of Lissoclinum differ little from those of Diplosoma. The cells of the terminal pad, however, do not show secretory drops or their precursors. Instead, there arises from the apex of each cell a fine strand or tuft of strands extending outwards and fusing with the substance of the common test (fig. 2C). This process is slightly different from that found in Diplosoma and somewhat resembles tunicization (see p. 254).

Didemnum maculosum

The colony of Didemnum is much more compact than that of Diplosoma or Lissoclinum. The common cloacal cavities are well-defined channels and there is no long strand of test extending below the zooids. The stolonic test vessels are like those of the two species already described but show a less marked tendency to pass downwards below the abdomen. Terminal bulbs are found as in the other forms. In zooids with buds that are still quite small, short stolonic vessels can be seen to originate from the epidermis posterior to the buds and close to the stomach (fig-3A).

FIG. 3.

The abdomen of A, Didemnum maculosum, and B, Trididemnum tenerum, showing the origin of stolonic vessels and buds.

FIG. 3.

The abdomen of A, Didemnum maculosum, and B, Trididemnum tenerum, showing the origin of stolonic vessels and buds.

Trididemnum tenerum

In this species the test is fairly solid and the common cloacal cavities not extensive. The stolonic test vessels stretch obliquely upwards from the zooid into the test (fig. 3B). They are almost straight and have large conspicuous terminal swellings. In this species also there is no extension of test as a strand below each zooid, a condition correlated with the lateral position of the vessels. The vessels originate from a point posterior to the buds and close to the stomach. The terminal bulbs examined did not show the method of test production.

The origin of the material of the ascidian test has interested many zoologists. In general it has been thought to originate in one or more of four ways:

  1. as a secretion of the epidermal cells;

  2. by the activity or transformation of mesenchyme cells which have passed through the epidermis;

  3. by transformation of the outer border of the epidermal cells in a process that Pérés (1948) named tunicization;

  4. by the outward passage of a substance through intercellular spaces in the epidermis (Saint Hilaire, 1931).

Pérés considered it unlikely that a substance passes through epidermal spaces. Indeed, it is doubtful if such spaces exist. Saint Hilaire and Pérés both noted cases of the production of test substance by immigrant mesenchyme cells, but this is apparently exceptional. The process of tunicization of epidermal cells occurs in the Didemnidae, but the most general method of test production in ascidians seems to be by epidermal secretion. In spite of this few workers have found satisfactory histological evidence of the process, although Pérés has described and illustrated it in the Polyclinidae. Amongst the Didemnidae tunicization is apparently the predominant process and Pérés found no clear evidence of epidermal secretion in the family. The terminal swellings of the stolonic vessels therefore provide one of the few clear cases of the secretion of test by epidermal cells. The stolonic test vessels of the Didemnidae have retained their ability to secrete tunicin, but this function is now entirely or largely concentrated in the specialized cells of the terminal swelling. It is uncertain to what extent mesenchyme cells migrate out of the vessels and take part in the production of the test.

The vessels either have lost the power of budding or never possessed it. This power is now confined to an area between the stolonic vessels and the muscular appendix. If it is true that, in the evolution of the Didemnidae, the stolonic vessels and the basal attachment of the longitudinal muscles have moved forward to their present position beside the abdomen, then it is probably also true that the area now concerned in budding was derived from the posterior end of the body of the ancestral form. In the clavelinid stock, which, as already suggested, may have given rise to the Didemnidae, the posterior end of the body is involved in budding of several kinds. The forward shift of the posterior end of the body, caused by reduction in the size of the zooid, may have carried the budding area to its present position near the oesophagus and led to pyloric budding.

A similar process starting from a polyclinid-type zooid is conceivable but less likely, as it would involve the much greater modification of a more highly specialized zooid. It is therefore suggested that the Didemnidae and the Polyclinidae both evolved from the clavelinid stock, the Didemnidae by reduction, and the Polyclinidae by elongation, of the zooid.

Berrill
,
N. J.
,
1935
.
Phil. Trans. Roy. Soc. B
,
225
,
327
.
Lahille
,
F.
,
1890
.
Recherches sur les tuniciers des côtes de France. Toulouse
.
Pérés
,
J.-M.
,
1948
.
Ann. Inst. Océan. Monaco, n.s
.
23
,
345
.
Saint
Hilaire
, K
.,
1931
.
Zool. Yahrb. Jena Anat
.,
54
,
435
.