Results

A. HISTOLOGICAL OBSERVATIONS: The body wall of I. olivacea when seen in sections varies between 30-70μ thick depending on the state of contraction of the animal. It is made up of 3 layers; an outer epithelial layer (ectoderm), an inner epithelial layer (endoderm), and a middle layer (mesogloea) (Pl. 1, Fig. 1).

Ectoderm: The ectoderm is composed mainly of supporting cells and gland cells. The supporting cells (Pl. 1, Fig. 2; Text Fig. 1e) are tall and slender, with a small (4-5μ × 2-3μ) nucleus which usually is placed about half way up the cell but which often occurs near the free surface. Frequently these cells are so slender as to appear thread-like. In such cases the nucleus bulges out from the cell. At the free surface the cells are usually expanded slightly and bear a short (3μ), flattened, tongue-like projection. At the mesogloea the cells taper away, but may have 2 or 3 short strands, or root-like processes. These features were noted from dissociated ectodermal tissue. It was found to be impossible to note all of these features in sections of body wall, as the cells were too long and thin and were often obliterated by dense packing of heavily staining gland cells. Many small oval nuclei are visible in sections, but it is difficult to assign them to any particular cell. The surface of the ectoderm appears ciliated when seen in section (Pl. 1, Figs. 1, 3, 5). However experiments with charcoal particles revealed no ciliary or flagellar current over the surface of the column. In fact only on the pharynx surface were the particles moved. When placed on pharynx tissue the charcoal particles moved rapidly towards the gastric cavity. In trypsin dissociation preparations no ciliary or flagellar movement could be identified on ectodermal cells, while cells of the endoderm showed vigorous flagellar movement.

Text Fig. 1 (Left): Diagrammatic representation of the column ectoderm as seen in a transverse section stained by the Mallory/Azan technique.

Scale measures 20μ.

a, type A gland cells; b, type B gland cells; c, type C gland cells; e, supporting cell; m, mesogloea; n, nucleus; p, projection; s, nerve cell.

Text Fig. 2 (Right): Nematocysts found in the column ectoderm in I. olivacea.

Scale measures 10μ.

a, atrichous anisorhiza; b, microbasic amastigophore; c, holotrichous isorhiza; d, capsule; e, butt; 1, lid; t, tube.

The gland cells in the ectoderm are numerous and often closely packed. In Mallory/Azan preparations three types of gland cell can clearly be distinguished on the column (Text Fig. 1). One type contains granules which stain orange red and are up to 1.5μ in diameter. These will be called type "A" cells. They are narrow (about 3μ wide) but often are wider (up to 7μ wide) at the surface, and they make up about 35% of gland cell numbers. A second type ("B" gland cells) contain granules which are about 0.7μ in diameter, and which stain a red-purple colour. The granules are very closely packed and stain so intensely that often individual granules are difficult to discern from their neighbours. These cells are 3-4μ wide with a tapering base, and account for about 40% of gland cell numbers. The third gland cell type (type "C") stains a pale blue colour. The cells are usually about 4-5μ wide throughout their length, and appear bulkier than the other two types. They contain pale staining oval granules about 2μ long (long axis). The periphery of the granules often colours more deeply than the centre, giving the cell contents the appearance of a coarse, irregular network. Near the mesogloea the network page 6 may be very coarse, and the granules in this case are not visible. About 20% of the gland cells are of this type.

Localised modifications of this ectodermal pattern occur over the column surface. At various sites oval or circular areas of about 0.1 mm. diameter can be seen with a binocular microscope, which are paler than the olive green colouration of the rest of the column. In sections these areas are seen to be ectodermal invaginations which differ histologically from adjacent areas in that the previously described gland cells are absent (Pl. 1, Fig. 5). In Mallory/Azan preparations two new gland cell types can be distinguished. One type contains orange staining granules with a diameter of about 0.5μ; the other holds deep blue-grey coloured material which may appear homogenous or as ill-defined granules 0.5-1.0μ in diameter. The bases of both types of cell are 3-5μ wide, and they open to the surface through long, slender necks. The ectodermal supporting cells are more clearly seen in these areas.

Dissociation techniques and phase contrast microscopy show the nematocysts present in the column body wall to be of three types (Text Fig. 2): microbasic amastigophores, holotrichous isorhizas, and atrichous anisorhizas (according to the classification of Weill, in Hyman 1940). In the unexploded state the microbasic amastigophores measure about 18μ × 2-3μ and are slightly curved. The holotrichous isorhizas measure 15μ × 3-4μ, and the atrichous anisorhizas 15μ × 7μ. The microbasic amastigophores and holotrichous isorhizas are the same width for most of their length, but the atrichous anisorhizas are ovoid.

Attempts at nerve staining with reduced methylene blue were unsuccessful. However, an ectodermal nerve net is visible in sections stained by Mallory/Azan (Pl. 1, Fig. 1N) and in iron haematoxylin preparations. An endodermal nerve net is also visible in these preparations. Both nerve nets are intraepithelial. Connections between the nerve nets through the mesogloea were not observed. These findings are in line with those of Bullock and Horridge (1965) concerning the nervous system of actinians.

Mesogloea: The middle body layer varies in thickness with the state of contraction of the animal, between about 10 and 30μ. It is reduced to about 3μ in thickness beneath the modified ectodermal areas described above. Both cells and fibres are present in this layer.

In section the cells (Pl. 1, Fig 1, arrow; Pl. 2, Fig. 3, arrow) are irregularly shaped, often elongated between bundles of fibres, and have round or elongate nuclei of about 3μ diameter or 6μ long respectively. The cytoplasm of the cells often closely invests the nucleus as a thin layer, but often is seen to be aggregated to one or other ends of the cell. Very fine granules (0.3μ diameter) which stain orange in the Mallory/Azan technique are sometimes seen in the cytoplasm. The cells frequently are located in spaces between bundles of fibres.

Staining with reduced methylene blue (Pantin, 1948) resulted in blue granules appearing in the cytoplasm of the mesogloea cells, often in a paranuclear position. Other similar sized granules appear to lie free between mesogloea fibres.

The fibres of the mesogloea stain intensely with the aniline dyes aniline blue (in the Mallory and Mallory/Azan technique, Pl. 1, Fig. 1) and fast green FCF (in the Wineera triple stain). In a well extended animal the fibres are arranged as bundles which appear to run mostly as page 7 alternate circular and longitudinal layers. In a contracted specimen this orderly arrangement is not so apparent. Fibres from different layers anastomose frequently. In transverse sections the layers of fibres immediately beneath the endoderm appear to be the thickest and most orderly in arrangement (Pl. 1, Fig. 6). In the outer region of mesogloea the fibres form a more or less loose network. Immediately beneath the ectoderm is a thin, dense line which stains blue in Mallory/Azan preparations. Between the mesogloeal fibres are spaces of varying size.

Endoderm: The inner epithelial layer is composed of epitheliomuscular cells (Pl. 1, Fig. 4) and gland cells. The former type are the most numerous, making up about 90% of the cells present.

The epitheliomuscular cells vary greatly in length and breadth according to the state of contraction of the animal. In a contracted animal the epithelial part of a cell may reach 100μ in length and in this case appears thread-like. This lengthening of the epithelial portion of the cell is complementary to and is caused by, a shortening of the basal muscle fibre. Conversely in a relaxed animal the epithelial part of the cell is about 30μ long and is more columnar; the base of the cell tapers for a short distance before fanning out (in one plane) to enclose the extended muscle fibre. The nucleus of these cells is ovoid in sections, measures about 6 × 3μ and is placed in the half of the cell closest to the coelenteron. This region of the cells is expanded slightly and bears a single flagellum at the end. The basal muscle fibres are ribbon-like structures which run in a cirmcumferential direction in the body wall of the column, making up the "circular muscles" of the animal. Once again, many of these features could be noted only by examination of dissociation preparations.

Large numbers of greenish algae are present in the epitheliomuscular cells.

The gland cells present in the endoderm colour differentially with the Mallory/Azan technique. One type, which contains closely packed bright red granules 0.7-1.0μ in diameter accounts for about 95% of the gland cells present. The other 5% of gland cells is made up by cells which stain blue. Indistinct granules 1.0μ in diameter are sometimes apparent in these cells, but most frequently the contents have a turbid appearance, similar to that of the Type C ectodermal cell.

B. HISTOCHEMICAL OBSERVATIONS: These results are summarised by tables 1 and 2.

Ectoderm: Histochemical results for the ectodermal supporting cells were gained by examining the areas of modified ectoderm described earlier, in which the supporting cells are especially numerous. In other areas of the column it is impossible to study the supporting cells because of the abundance of gland cells. The classification of gland cells based on the uptake of dyes in various histological techniques is found to be too simplified when histochemical methods are applied: The type "A", "B", page 8 and "C" cells retain their individuality, but several other types are recognisable also. Firstly, one type of cell which contains granules of similar size to those of cell type "B". These cells, which will be called "B2" cells, are much more slender than the type "B" cells, and are clearly distinguished by their histochemical reactions (see Tables 1 and 2). A second new type of gland cell (type "D") is composed of very fine (0.5μ)

Table 1: Reaction of the body wall constituents of I. olivacea to tests for carbohydrates.

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Table 2: Reactions of the body wall constituents of I. olivacea to tests for protein, lipid, and Nucleic acids.

page 10 granules which appear to be of two kinds, judging by their staining reactions. These cells are best seen at the base of the ectoderm. They open to the surface by very fine cell necks. A third type of cell (type "E") is present, and is distinguishable on histochemical grounds, although it resembles the type "C" cell, in morphology. This type of gland cell opens to the surface by a narrow duct. The histochemical reactions of the various gland cells are summarised in tables 1 and 2.

As mentioned earlier the patches of modified ectoderm scattered over the column surface possess two types of gland cells, one which stains orange red with the Mallory/Azan method and one which stains blue. These also show different and distinctive histochemical reactions. The former colour with naphthol yellow S, and the latter colour red in the PAS technique. When pepsin digestion precedes the Mowry colloidal iron-PAS-naphthol yellow S procedure, bright blue structures appear in these areas of ectoderm at the level of the nerve net. They are often stellate in appearance (Pl. 2, Fig. 4, arrows) and do not approach the size of the gland cells. They are not connected to the surface of the ectoderm.

Mesogloea: Many of the cells of the mesogloea contain diastase resistant PAS positive granules, and naphthol yellow S positive granules. The cytoplasm of many was also shown to colour moderately strongly with pyronin (Pl. 2, Fig. 3, arrows), and RNase treatment removed the stain. A strong positive reaction was obtained for lipid in these cells; the lipid is present as very small globules scattered throughout the cytoplasm. Pyridine extraction prevents the staining with oil red 0.

The mesogloeal fibres gave a positive reaction in the PAS test, and also stained with naphthol yellow S. After sulphation and toluidine blue staining they coloured purple, and this colour persisted in DePeX mounted sections. The fibres immediately outside the muscles stained moderately strongly for reticulin (Pl. 2, Fig. 2) as did a very thin layer immediately beneath the ectoderm.

Endoderm: The gland cells containing granules which colour red with Mallory/Azan (type "1" cells) colour with naphthol yellow S (Pl. 3, Fig. 1, P). The ones which stain blue with the Mallory/Azan method are distinguishable histochemically into two types: One type (type "2" cells) stain both with the PAS procedure (Pl. 3, Fig. 1, arrows) and with the Mowry colloidal iron method, and when these methods are used in combination these cells appear deep purple. Another type of gland cell (type 3) stains intensely with the Mowry colloidal iron method but is PAS negative (Pl. 3, Fig. 2, arrows).