Discussion

The Epidermis: In a previous paper (Wineera, 1969) it was stated that unicellular basiphil glands occur scattered throughout the epidermis. From the results of the present study it appears, however, that the basiphil secretions which were described in the epidermis are in reality the thin gland cell "necks" described by other workers (Hyman, 1959; Pedersen, 1963 as belonging to the subepidermal basiphil glands.

The epidermal "channels", and the cavities, or spaces between the bases of epidermal cells are interesting features. However, the relationship between these structures cannot be decided at present. Török & Röhlich (1959) state that planarian epidermal cells are attached to each other only superficially, and are surrounded by intercellular spaces of varying size, and Skaer (1961) and Pedersen (1961a) have described fluid filled cavities between the "basement membrane" and the bases of epidermal cells in fresh water triclads. Skaer states that the cavities appear to form a continuous system ramifying above the basement membrane, and suggests that their function may be hydrostatic or associated with the extrusion of granular secretion products through the epidermal cells.

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The present study supports the view of Wineera (1969) that the "basement membrane" as described in triclads is not a true basement membrane. That is, it is not a basal lamina (Fawcett, 1966). Plate 1, Figures 2-4 show two components in the "basement membrane": One is the thin dense line immediately beneath the epidermis, and the other is the thicker, less dense layer beneath the thinner one. It seems probable that the former represents the true basement membrane (basal lamina) of the epidermis or at least approaches it more closely than any other structures visible with the light microscope, while the thicker layer is the "connective tissue layer" or "connective layer" as previously described (Wineera, 1969).

The Muscles and Parenchyma: The muscle fibres of P. stephensoni appear to resemble closely those described by Skaer (1961) for a fresh water triclad. In both instances the fibres give off fine processes along their length, and the nuclei may be situated some distance from the contractile part of the cell. According to Skaer the fine processes consist of aggregates of mitochondria bounded by the plasma membrane of the cells. It is probable that these processes account for some of the fibres described as connective tissue fibres (Wineera, 1969).

Three recent studies on turbellarians give slightly different pictures regarding the parenchyma and the occurrence within this tissue of connective tissue fibres. Two of these studies were carried out on triclads: In one (Skaer, 1961) no mention is made of the occurrence of fibres in the parenchyma; in the other (Pedersen, 1961a) it is stated that the cells of the parenchyma are closely packed together leaving a gap only a few hundred angstroms wide between two opposing cell membranes. Occasionally this gap is widened, especially around muscle cells, and filamentous material is often found in this narrow intercellular space. The third paper is a study of a marine polyclad turbellarian (MacRae, 1965), in which is shown the occurrence between muscle fibres and around muscle fibre bundles of extracellular fibrillae, presumed by MacRae to be an early form of collagen. Measurements of the regions of these fibrillae from MacRae's electron micrographs show them to be from 800-2,800 A in diameter. These larger regions possibly would be visible with the light microscope as thin fibres. It is possible that the connective tissue fibres associated with muscles as described by Wineera (1969) may be regions of extracellular fibrillae similar to those described by MacRae. Pedersen (1961a, p.597) suggests that the filaments (extracellular fibrillae of MacRae) occurring between muscle cells might be identical to those occurring in the basement membrane (Pedersen's "basement membrane" is equivalent to the basement membrane plus the connective layer of the present study) and says that "... there often seems to be a continuity between the subepidermal basement membrane and the intercellular space around the outer circular muscle cells." This same feature is recorded by Wineera (1969), and is seen in the present study. However, the present study has shown that the terminology used by Pedersen to describe this feature should be modified: It is not the basement membrane but the connective layer which projects between the muscles (Pl. 1, Fig. 4, arrow).

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None of these papers mentions the origin or insertion of these muscle fibres or fibre bundles. It is axiomatic that muscle fibres or fibre bundles have to be attached in at least two places some distance apart in order to effect movement of body parts. In higher animals it is connective tissue which provides anchorage. MacRae (1965) suggests that a parallel exists between the filamentous material surrounding muscle fibres in the planarians and the endomysium round the muscle fibres of higher animals; and the material round planarian muscle fibre bundles and perimysium in higher animals. In the present study fine processes arising from muscle fibres are seen to join the connective layer (i.e. the basement membrane of other workers) (Pl. 1, Figs. 3, 4, arrows). It is suggested that these processes are analagous to tendons and similar structures of higher animals serving as attachments to the "connective tissue skeleton" supplied by the thick connective layer ("basement membrane").

The cells in the parenchyma which show curvilinear patterns in their cytoplasm are particularly interesting. At first sight these cells bring to mind the picture of a cell with a highly developed granular endoplasmic reticulum in which the cisternae and their attached ribosomes are arranged in parallel arrays. This specific arrangement of granular reticulum is characteristic of cells elaborating protein rich secretions for export from the cell (Mercer, 1961; Fawcett, 1966). Garnier (1897, 1899; quoted in Haguenau, 1958) noted the occurrence of basiphil "filaments" or "rods" in the basal cytoplasm of sumaxillary salivary gland cells. For these structures he coined the term "ergastoplasm" (Greek "ergazomai"—to elaborate and transform) for he believed in their importance in the secretory activity of the cells. The electron microscope reveals that "ergastoplasm" consists of the particular arrangement of cisternae and ribsomes described above. Bloom and Fawcett (1966, p.10) show an electron micrograph of pancreas at a magnification of 2,700 x. The appearance in this micrograph of the granular reticulum is very similar to that of the strands of basiphil cytoplasm of these parenchyma cells of P. stephensoni. Moreover, measurements of the cisternae plus attached ribsomes from Bloom & Fawcett's micrograph show them to be in excess of 0.2μ wide. This would make them visible (theoretically) in the light microscope, providing the sections were thin enough and the ribsomes were present in sufficient quantity to confer the needed basiphilia on the cytoplasm around them. It is seen from the micrograph of Bloom & Fawcett that at this magnification the lumen of each cistern is obliterated, so that each cistern plus its attached ribsomes appears as a single dense line. It is possible, then, that the "strands" seen in these cells in P. stephensoni may be dense lines representing cisternae plus ribsomes of a very well developed and regularly arranged granular endoplasmic reticulum. However, for the present the term "ergastoplasm" must be used to describe these structures since this term refers to cytoplasmic features visible with the light microscope. Pedersen (1963) shows that a highly developed granular reticulum occurs in subepidermal eosinophil gland cells in fresh water triclads in the early stages of the secretion cycle. Measurements from an electron micrograph published by Pedersen of such a cell show that the cisternae plus ribsomes of the reticulum range from 0.1 to 0.35μ wide. It is, then, possible that in the present study the cells in which the ergastoplasm occurs are eosinophil gland cells at an early page 7 stage of secretion. The cells of similar size and staining reaction which contain pale granules would then possibly be eosinophil gland cells at a later stage of the secretion cycle containing partly formed secretion granules. It is noticeable that, as has already been mentioned, the eosinophil secretion granules stain very faintly or not at all with MBAII.

Two other possible explanations of these cells must be noted. The first explanation is that the strands may represent an extremely well developed Golgi apparatus. However, the extent of the strands in relation to the size of the cells tends to negate this suggestion; also it is not understood what would bind the stain if these strands were part of a Golgi apparatus. A second "explanation" is that the "strands" are manifestations of some other cellular constituent, such as microtubules, or are arrangements of particular substances. This is a question which probably could be solved with the electron microscope.

Pigment: In a previous study (Wineera, 1969) it was concluded that the pigment granules of P. stephensoni were distributed at the sites of connective tissue fibres particularly where these fibres course between the muscle fibres of the dorsal body wall. This view can no longer be held.

It is now clear from this present study that pigment is present in distinct pigment cells which are located in the parenchyma. Pigment cells "in vitro" are rounded in shape (Pl. 2, Fig. 4A, 4B). However, the shape of the cells "in vivo" probably is more stellate, and they probably ramify between muscle cells in order to appear as they do in sections. Support for this view is given by Needham (1965, p.210) who states that pigment in Polycelis nigra (a fresh water triclad) occurs in "fairly orthodox asteroid cells". However, Skaer (1961) states that Polycelis pigment occurs within muscle cells and nerve cells.