Zoology Publications from Victoria University of Wellington—Nos. 58 to 61
A. HISTOLOGICAL OBSERVATIONS: The body wall of S. tenella is made up of two epithelial layers, an outer ectoderm and an inner endoderm, which are separated by a thin layer of mesogloea (Pl. 1, Figs 3, 5). Three morphologically distinct regions can be recognized in the sagittally sectioned hydranth (Pl. 1, Fig. 1). These are a basal region, a middle region, and an apical region. The cells of the body wall layers have characteristic features in these different regions.
ECTODERM. The ectoderm of the hydranth varies from a pavement to a columnar epithelium. In an extended animal the tentacle ectoderm is a thin, pavement-like epithelium, while the ectoderm of the body of the hydranth is more cuboidal. In a contracted specimen the tentacle ectoderm is more cuboidal, and that of the body of the hydranth often becomes somewhat columnar. At the mouth there is a distinct ectodermal/endodermal junction, the cuboidal ectodermal cells giving way to the tall columnar cells of the endoderm.
When seen in section the ectoderm is covered by a thin (0.3μ) layer which grades into the perisarc at the hydranth base. This layer stains densely with iron haematoxylin, and with aniline blue in the Mallory/Azan technique (Pl. 2, Fig. 1, C). In a contracted animal it is extensively folded (Pl. 4, Fig. 4, C). Sometimes it is seen in a tangled mass which has been separated from the ectoderm (Pl. 1, Fig. 4, C). At the basal region of the hydranth this thin layer becomes covered by a thicker but less densely staining layer which often seems loosely applied to it. These layers then grade rapidly into the perisarc of the hydrocaulus which has two components: an inner layer which stains densely with aniline blue in the Mallory/Azan technique and which is continuous with the thin layer present over the hydranth, and an outer layer which stains lightly and is continuous with the thicker layer present in the basal hydranth region described above (Pl. 2, Fig. 5). These observations apply only to the annulated region of perisarc immediately beneath the hydranth.
The most numerous cell type in the ectoderm is the epitheliomuscular cell. Cells of this type vary in shape from pavement to columnar epithelial cells (as mentioned above) and each possesses a single muscle fibre in its basal portion. The muscle fibres run longitudinally, constituting the longitudinal musculature of the hydranth. They are best seen in iron haematoxylin and Mallory/Azan preparation (Pl. 3, Fig. 1A, MY). When seen in FAA fixed sections the cells possess a large (5μ diameter) circular nucleus situated centrally. A prominent densely staining nucleolus is present in each nucleus. At the hydranth base the ectoderm becomes thickened, and sometimes appears more than one cell thick. However on close examination the epitheliomuscular cells appear to retain their connection with the mesogloea. In this region the cells stain more heavily with basic dyes such as haematoxylin, and with azocarmine in the Mallory/Azan technique.page 5
Nerve cells are also present in the ectoderm. In vital methylene blue preparations they are most numerous in the hydrocaulus, but also occur scattered over the hydranth. The nerve cells are mostly spindle shaped and bipolar, sometimes multipolar, and are situated at the base of the ectoderm just above the mesogloea. They are visible in iron haematoxylin and Mallory/Azan preparations by virtue of their basiphil cytoplasm, and their orientation at right angles to the ectodermal cells (Pl. 2, Fig. 1). They are most clearly seen in Formol/calcium fixed material. Sensory cells, which extend from the level of the nerve cells to the free surface of the ectoderm are also present, and possess fine cilia-like processes (Pl. 2, Fig. 2, S). Where these processes reach the surface the thin outer layer of the ectoderm is absent. Sensory processes also reach the ectodermal surface directly from the nerve cells. The nerve cell processes of the hydrocaulus run between the bases of the ectodermal cells. It was not possible to discover the position of nerve cell processes in relation to epitheliomuscular cells for the hydranth, nor was it possible to determine whether the sensory cells or the sensory processes of nerve cells reach the ectodermal surface by passing through or between ectodermal cells. Nerve cell processes are occasionally seen crossing the mesogloea to endodermal cells (Pl. 1, Fig. 7, N). Another cell type is visible in sections. These cells are small, approximately 5μ diameter, with cytoplasm evenly distributed around a nucleus of approximately 3μ diameter. A nucleolus is present. Cells of this type are found singly or in pairs between the bases of ectodermal cells (Pl. 1, Fig. 5). They are easily distinguished from nerve cells by their small size, and lack of polarity.
Nematocysts (all of which are ectodermal) occur predominantly in the swollen heads of the scattered capitate tentacles. However they also occur in the tentacle shaft, scattered over the hydranth body, and in the hydrocaulus. In this last region they are numerous (Pl. 3, Fig. 4). Three types of nematocyst have been positively identified in dissociation preparations. These are stenoteles, atrichous isorhizas (according to the classification of Weill, cited in Hyman (1940), and a third type (Pl. 4, Fig. 6 B) resembling a stenotele without stylets. A fourth type (PI. 4, Fig. 6A) is less frequently seen. This resembles type 3, but does not show the same curved structures within the capsule. Three size ranges of the first type occur, in which the capsule length × width is 8μ × 5μ, 12μ × 9μ, and 16μ × 13μ respectively. The second type of nematocyst measures 5μ × 3μ, and the third and four types 16μ. × 13μ. Developing nemotocysts are seen in the stolon, and on the hydranth body. They have not been seen on the tentacles.
MESOGLOEA. The mesogloea is acellular, and in animals anaesthetized before fixation it appears in sections as a thin line. It stains intensely with aniline blue in the Mallory/Azan technique. It is more easily studied in non-anaesthetized animals, for contraction of the animals during fixation thickens the mesogloeal layer. In such animals fibres are visible in the mesogloea (Pl. 3, Fig. 1A and 1B). These appear to be arranged longitudinally beneath the ectodermal muscle layer, and circularly beneath the endodermal muscle fibres. However the fibres could be followed for only a short distance, so it is possible that they are cut obliquely and have a different orientation. Between these coarse mesogloeal fibres there often appears to be a much finer fibre network. In page 6 the longitudinal section of a contracted animal, the mesogloeal layer immediately beneath the endoderm is thrown into folds, along with the endodermal epithelium (Pl. 4, Fig. 5, M).
ENDODERM. This layer consists of epitheliomuscular cells, gland cells, and nerve cells. The former are columnar epithelial cells which possess a basal muscle fibre, and a single flagellum at their free surface (Pl. 1, Fig. 8). The muscle fibres are orientated circularly constituting the circular muscles of the hydranth body wall. In the basal and middle regions of the hydranth the epitheliomuscular cells are usually large (9μ × 20μ in sagittal section) and often subtend two ectodermal epithelial cells at their base. In sections of FAA fixed specimens the basal half of the cell appears empty (Pl. 1, Fig. 3; Pl. 4, Fig. 5), with the cytoplasm and nucleus placed in the apical (distal) half. The nuclei of these cells measure 4-5μ diameter and appear circular or near-circular in section. Each possesses a prominent nucleolus. In the apical region of the hydranth the epitheliomuscular cells become more slender.
If an animal is sectioned after a period of starvation, the apical region of the cells contains vacuoles in which are small dark bodies (Pl. 4, Fig. 5). Flagella are clearly visible. When an animal is sectioned 12-18 hrs. after feeding these cells are packed with eosinophil globules 0.5 - 2.0μ in diameter. Many of the globules are aggregated inside vacuoles. They are very well shown in preparations fixed in Baker's formol/calcium (Pl. 2, Fig. 1). Flagella are not seen, in this instance, as commonly as when the animal is fixed and sectioned before feeding. Vacuoles containing similar staining globules are also seen in the tentacle endodermal cells (Pl. 4, Fig. 3).
Scattered throughout the endoderm of the middle hydranth region are secretory cells (Pl. 3, Fig. 2, D). They are tall and slender (15-20μ × 8μ) in section, with a round nucleus 4μ in diameter which is centrally placed. A prominent nucleolus is present in FAA fixed preparations. The basal cytoplasmic regions stain intensely with basiphil dyes such as haematoxylin, and with azocarmine in Mallory/Azan preparations. The apical region of the cells contains eosinophil granules approximately 1.5μ in diameter. After a period of starvation most of these secretory cells are packed full with eosinophil granules and little basiphil cytoplasm is evident. However 12-18 hrs. after feeding fewer eosinophil granules are present in the cells, which now stain heavily with basic dyes.
Gland cells are numerous in the apical hydranth region (Pl. 1, Fig. 2, G). They are tall and slender (15μ × 8μ) often with a slightly swollen distal end. Two types are recognizable histologically: One type possesses moderately eosinophil cytoplasm which is finely granular. The other type stains with aniline blue in the Mallory/Azan technique. Cells of this second type also appear finely granular, but sometimes a network of densely staining material is present. In an animal fixed with FAA without prior anaesthetization, the endoderm is thrown into villi-like radial folds. These are very prominent in the apical region of the hydranth (Pl. 1, Fig. 2). They run longitudinally down the endoderm becoming less prominent in the middle region and unrecognizable in the basal region of the polyp. The glands of the apical region of the polyp are disposed around the perimeter of the folds. In animals anaesthetized before fixation the folds are much less prominent.page 7 page 8
Table 2: Reactions of the body wall constituents of S. tenella to tests for RNA, protein, and lipid.
The tentacles are solid, with a core of endodermal cells. These are all epitheliomuscular cells which do not bear a flagellum. A longitudinal section of a tentacle shows two layers of endodermal cells in close contact. The cytoplasm and nucleus of the cells of each layer are distally placed and thus are in close proximity (Pl. 4, Fig 1, EN). In transverse section the endodermal cells are seen to be radially arranged, with the cytoplasm and nucleus of each cell close to the centre of the tentacle (Pl. 4, Fig. 2).
B. HISTOCHEMICAL OBSERVATIONS: Histochemical results are summarised in tables 1 and 2. Features not able to be expressed in the tables or those not readily seen from the tables are described below.
ECTODERM. 12-18 hrs after feeding many of the epitheliomuscular cells of the ectoderm show an intensely PAS positive substance in the form of irregularly shaped bodies. These are removed by digestion with diastase for 30 mins. (Pl. 2, Figs 4A, 4B), and in table 1 are denoted as "PAS +++ INC.". Others of the same type of cell contain very fine granules which are also intensely PAS positive (Pl. 1, Fig. 6). These granules are denoted as "PAS +++ FG" in table 1. They are resistant to diastase digestion, but are removed by pepsin digestion. Their presence seems unaffected by the feeding state of the animal.
At the base of the hydranth, the ectodermal cells contain coarse granules which stain intensely with Mowry's colloidal iron reagent. This is not shown in table 1 (see Pl. 5, Figs. 1A and 1B).
The nerve cells stain strongly with pyronin in the methyl green/pyronin technique. This is prevented by digestion with RNase.
The small round cells found at the base of the ectoderm display moderate pyroninophilia which is removed by RNase digestion.
MESOGLOEA. In the PAS/AB/NYS test before pepsin digestion the mesogloea colours an intense red with here and there patches of deep blue (Pl. 4, Figs. 4A, 4B); however this appearance is reversed after pepsin digestion, that is, the mesogloea appears blue with patches of red.
ENDODERM. Intensely PAS positive structures which are diastase labile are found in the endodermal epitheliomuscular cells a short time after feeding. This is not shown in table 1. Animals sectioned 12-18 hrs. after feeding with Artemia show fragments of Artemia exoskeleton, of various sizes, within endodermal cells of the basal and middle hydranth regions. These fragments stain with the PAS and AB techniques.
Three types of gland cell can be distinguished histochemically in the apical hydranth region. One type (type 1) is only found immediately surrounding the mouth. It is very finely granular and intensely PAS positive. Another type (type 2) extends from a region just below the mouth throughout the apical region of the hydranth. It mostly appears as a "network" structure and stains moderately strongly with the PAS test but intensely with the AB and Mowry colloidal iron tests (Pl. 3, Figs. 3A, 3B). The third gland cell type stains strongly with NYS. It is also finely granular, and is found around the mouth region. Types 1 and 2 appear to correspond to the gland cells which stain with aniline blue in Mallory/Azan preparations, while type 3 corresponds to eosinophil glands described earlier.