Hydrobiologia 347: 15–24, 1997. 15c 1997 Kluwer Academic Publishers. Printed in Belgium.

Ultrastructural features of the epidermis of the planarianArtioposthia triangulata (Dendy)

Catherine McGee1, Ian Fairweather1;� & Rod P. Blackshaw2

1School of Biology and Biochemistry, The Queen’s University of Belfast, Belfast, BT7 1NN, Northern Ireland(�Author for correspondence)2Department of Agriculture and Food Studies, University of Plymouth, Devon, TQ12 6NQ, England

Received 25 April 1996; in revised form 23 January 1997; accepted 13 February 1997

Key words: Turbellaria, Artioposthia triangulata, ultrastructure, epidermis, secretions

Abstract

The epidermis of the land planarian Arthioposthia triangulata was examined by scanning and transmission electronmicroscopy. This investigation revealed that the flatworm was covered entirely with cilia and was especially denselypopulated on the ventral surface. In all regions the epidermis consisted of a one-layered columnar epithelium restingon a prominent basement membrane, but lacking a terminal web. Various secretions were found in the epidermistogether with epidermal rhabdoids. Below the basement membrane other secretory material was visible and thisincluded the cytoplasmic lamellated granules and adenal rhabdites. The basement membrane consisted of fibrilswith a beaded appearance and these were arranged parallel to the epidermal layer but did not display cross-banding.The secretory cells above and below the basement membrane were compared and their products characterized onthe basis of shape, size and location. Their possible function is discussed.

Introduction

The epidermis of turbellarians consists of a single lay-er of epithelial cells and is separated from the musclelayers by a basement membrane. Turbellarians do notpossess a cuticle and the epidermis is entirely ciliated.This provides their means of locomotion, by ciliarygliding. In some species, however, reduction of ciliais common: for example, in some of the interstitialsand-dwelling species and some of the temnocephali-dans the dorsal cilia may be reduced or absent leavinga ventral ciliated creeping sole (Rieger et al., 1991).Even with such a simple epidermis there is consid-erable variation among species. Some species havedeveloped insunken epithelial cells with the nuclei ofthese cells located among or below the muscle lay-ers. Some have developed a syncytial epidermis, whileothers have elaborate intracellular cytoskeletal struc-tures and extracellular matrices such as a basementmembrane. One of the main sources of variation inthe turbellarian epidermis is the difference in appear-

ance, distribution and abundance of gland cells such asmucoid cells and rhabdites (Rieger et al. 1991).

The epidermis of most turbellarians rests on anextracellular matrix, which takes the form of a base-ment membrane, whose main functions are to providemechanical support for the epithelial cells, and to serveas an attachment site for the body wall musculature.In general, the degree of development of the basementmembrane corresponds to the size of the organism, andthe triclads fall into the category of those possessinga true basement membrane (Pedersen, 1966; Ehlers,1985).

There is considerable variation in the glandularsecretions of turbellarians. The gland types have beenidentified largely on morphological grounds, with lit-tle known about their composition or function. Mostof the gland cells lie beneath the epidermal layer in theparenchyma and their necks project through the musclelayers and basement membrane to penetrate the epider-mal cells directly or pass between them and eventuallymake their way to the epidermal surface.The epidermisof aquatic turbellarians has been described extensive-

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ly, but relatively little is known about the ultrastructureand general characteristics of the epidermis of landplanarians (Storch & Abraham, 1972; Bedini & Papi,1974; Curtis et al., 1983; Winsor, 1983).

Artioposthia triangulata (Dendy, 1895) (Order Tri-cladida; Suborder Terricola) is a native land planarianof New Zealand, and has attracted much attention inIreland and Britain due to its predation on earthwormspecies. It has been recorded in Ireland since 1963(Willis & Edwards, 1977) having been introduced,most probably, by the plant trade. Since that time it hasspread throughout Ireland, to Scotland and to England(Blackshaw & Stewart, 1992). A. triangulata feedson all earthworm species, with the surface-dwellingspecies, such as Lumbricus terrestris, being partic-ularly vulnerable. Due to this, A. triangulata posesa potential long-term problem for earthworm popula-tions (Blackshaw, 1992; Blackshaw & Stewart, 1992).Work to date has focused on the distribution,spread andbasic biology of A. triangulata (Blackshaw & Stewart,1992) and only a small amount of ultrastructural workhas been done (McGee et al., 1996).

This study has been undertaken to examine thecytological features of the epidermis and, in partic-ular, to examine the various secretions produced bythe epidermis and associated gland cells. Scanning andtransmission electron microscopy have been employedto observe the outer surface and the ultrastructure ofthe epidermis. Among the various secretions presentin the epidermis of A. triangulata are rhabdites, whichare very characteristic of turbellarians (Hyman, 1951).They are produced both in the epidermis and beneaththe basement membrane, but both are secreted at theapical surface. The rhabdites are described in a separatepaper (McGee et al., 1996).

Materials and methods

Specimens of Artioposthia triangulata were collectedfrom different areas in County Down and kept in thelaboratory at 5 �C, in a sealed container lined withmoist filter or tissue paper until required.

For transmission electron microscopy (TEM), theflatworms were flat-fixed in 4% (w/v) glutaraldehydein 0.1 M sodium cacodylate buffer (pH 7.4) containing3% (w/v) sucrose for 0.5 h and 1 mm-square piecesof tissue were dissected from different regions of theworm. The pieces of tissue were left overnight at 4 �Cin 4% (w/v) glutaraldehyde in 0.1 M sodium cacody-late buffer (pH 7.4) containing 3% (w/v) sucrose. They

were then buffer-washed in 0.1 M sodium cacodylatebuffer (pH 7.4) for approximately 24 h at 4 �C andpostfixed in 0.5% aqueous osmium tetroxide for 1 hat room temperature. Following this, the tissue pieceswere washed in the buffer solution again and dehy-drated through a graded series of alcohols to propyleneoxide and embedded in Polarbed 812 Epon resin (AgarScientific Limited, Stansted, UK).

Initially, sections (1 �m in thickness) were cut withglass knives using a Reichert UM4 Ultramicrotome,and stained with 1% (w/v) Toluidine Blue in 2% (w/v)aqueous sodium tetraborate for light microscope exam-ination, to show the general morphology of the wormand to ensure the presence of the epidermis in the tissuesection. Ultrathin sections were then cut and double-stained with alcoholic uranyl acetate (5 min) and aque-ous lead citrate (8 min) (Reynolds, 1963). The sectionswere examined using a Jeol 100-CX transmission elec-tron microscope operated at 100 kV.

In addition, some pieces of the flatworm werestained to show acid phosphatase activity (Gomori leadmethod) at the TEM level (Pearse, 1972). The piecesof tissue were cut and fixed as above and incubated at37 �C in a substrate medium adjusted to pH 5.0 for 1 h.They were then washed in 0.1 M sodium cacodylatebuffer (pH 7.4) for 2–3 h, postfixed in 0.5% aqueousosmium tetroxide for 1 h at room temperature, buffer-washed again, dehydrated through a graded series ofalcohols to propylene oxide and embedded in Polarbed812 Epon resin.

For scanning electron microscopy (SEM), the flat-worms were immobilised in 4% (w/v) glutaraldehydefor 15 min. and dissected into six pieces. Each piecewas fixed in an aqueous solution containing 3 parts 4%(w/v) glutaraldehyde and 1 part 0.5% osmium tetroxidefor 4 h at 4 �C. They were washed thoroughly in severalchanges of deionised water and dehydrated through agraded acetone series. Tissue pieces were then dried inCO2 using an EMSCOPE CPD 750 critical point dry-er (Watford, England), mounted on aluminium stubsusing double-sided sticky tape and coated with a 50 nmlayer of gold/palladium in an EMITECH K550 sputter-coater. Specimens were examined in an ISI ABT 55scanning electron microscope operated at 10 kV.

For histochemistry, the flatworms were fixed in 4%(w/v) glutaraldehyde, cut into a number of pieces, andleft overnight at 4 �C in 4% (w/v) glutaraldehyde in0.1 M sodium cacodylate buffer (pH 7.4) containing3% (w/v) sucrose. They were then buffer-washed in0.1 M sodium cacodylate buffer (pH 7.4) for approx-imately 24 h at 4 �C, dehydrated through a graded

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ethanol series and embedded in JB4 plastic resin (AgarAids, Cambridge, England). Sections of 3 �m in thick-ness were cut on a pyramitome. For examination, sec-tions were stained with haematoxylin and eosin forgeneral morphology; toluidine blue in 2% (w/v) sodi-um tetraborate for tissue metachromasia; periodic acid-Schiff (PAS), with amylase control, for carbohydrates;Alcian blue, with varying pH values as a control, foracid mucopolysaccharides; chromotrope 2R for basicproteins and for acid phosphatase activity, with omis-sion of substrate serving as a control (by the Gomorilead method) (Bancroft & Stevens, 1977).

Results

A. triangulata possesses a single-layered ciliated epi-dermis, is devoid of a creeping sole and moves upon itsentire ventral surface by means of muscle contraction,mucus production and cilia. SEM micrographs revealthat the cilia are more densely populated on the ventral(Figure 1) than the dorsal (Figure 2) surface of A. tri-angulata, and the cilia are especially dense around thepharyngeal opening. However, there is no differencein length of the cilia between the ventral and dorsalsurfaces.

From the TEM micrographs it can be seen that themicrotubules of the cilia have a distinct 9 + 2 arrange-ment. Ciliated sensory receptors are dispersed alongthe surface of the epidermis. They display a prominentstriated rootlet and ladder-like septate desmosomes areevident on each side of the receptor joining it to the sur-rounding epithelial cells (Figure 3). The epidermal lay-er is 27�m in width and rests on a basement membrane4.5 �m wide. The basement membrane is composedof fibrils embedded in a homogeneous substance and,when magnified, the basement membrane has a beadedappearance. The fibrils do not have a regular appear-ance but are arranged in all directions and there appearto be longitudinal and circular fibres mixed together(Figure 4). A. triangulata does not possess a terminalweb.

Within the epithelial layer, several types of secre-tion are present and they have their origin either in theepidermis itself or in gland cells in the parenchyma.Each epidermal cell contains a round nucleus with aruffled surface which is usually situated in the mid-dle to lower half of the epidermal cell and is usually10–14 �m in diameter. The nuclei are surrounded bysmall Golgi complexes, mitochondria (0.25–0.5 �min diameter), dispersed cisternae of granular endoplas-

mic reticulum (GER) and various types of secretion(Figure 5).

In total, four different types of secretion have beenidentified in the epidermis of A. triangulata.

Type IType I secretory vesicles are small and irregular inshape and may appear empty. This secretion is pro-duced by the epidermal cells themselves and is pack-aged by the Golgi complex. This type of secretionis found in all epidermal cells. The vesicles do notincrease in size and, unlike Type III (see below), theydo not aggregate but move about individually in thecytoplasm of the cells (Figure 6).

Type IIThis secretion has the appearance of small, round, oftenempty-looking vesicles (approximately 0.2–0.4 �min diameter) contained inside a gland. Dependingon the plane of section, the contents of the vesiclesmay appear homogeneous. This secretion is producedbeneath the basement membrane in a specialized secre-tory cell containing a well-developed network of GER.The nuclei are round and contain 1–2 nucleoli. Fol-lowing production by the Golgi complex (Figures 7and 8), the vesicles aggregate in ‘packets’ (Figure 9).The ‘packets’ then aggregate in the neck of the cell(Figure 10). Via the gland cell neck, they make theirway through the muscle layer and basement membraneto the epidermal layer. The gland cell neck passesbetween the epidermal cells (Figure 11) and joins viaseptate desmosomes to the outer epithelium and thesecretion is released to the exterior.

Type IIILamellated cytoplasmic granules are the third type ofsecretion present in the epidermal layer of A. triangu-lata. They are produced in secretory cell bodies in theparenchyma, beneath the muscle layers (Figure 12).The nucleus of the secretory cell is smaller in compar-ison with other secretory cells (4 �m in diameter), butthe secretory cell is packed with a regularly arrangedand highly developed system of GER cisternae withswollen tips, especially when the cell is active in pro-ducing the cytoplasmic granules. Later, when the gran-ules have been formed, the GER disappears. There area large number of mitochondria present, multiple Gol-gi complexes and free ribosomes dispersed around thenucleus. When the granules are being formed, theyappear to be laid down in a manner which gives them

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Figure 1. Scanning electron micrograph (SEM) showing the distribution of cilia on the ventral surface of A. triangulata. (Scale = 1 �m)Figure 2. SEM of the distribution of cilia on the dorsal surface of A. triangulata. (Scale = 1 �m)Figure 3. Transmission electron micrograph (TEM) showing a portion of the apical region of a sensory receptor in the epidermal layer. Note thestriated rootlet (sr) and the septate desmosomes (arrow) joining the receptor to the surrounding epithelial cells. (Scale = 0.1 �m)Figure 4. TEM of the basement membrane (BM) situated just above the muscle layer (M). Lamellated cytoplasmic granules (Type III secretion)(arrow) can be seen passing through the basement membrane via gland cell necks to open into the base of an epidermal cell. (Scale = 0. 5 �m)Figure 5. Epidermal cell contains a round nucleus (N) with a ruffled outline. The cytoplasm contains rhabdoid, Type I (small arrow) and Type III(large arrow) secretions making their way to the ciliated surface. (Scale = 1 �m)Figure 6. TEM of Type I secretion (arrows), which is irregular in shape with dense contents. The vesicles are scattered throughout the epidermalcell and are especially abundant below the apical surface. Also visible are the epidermal rhabdoids (ER). (Scale = 0.25 �m)

their distinct banding pattern. They measure approxi-mately 1.2 �m in diameter but they do not appear to beindividually bound by a membrane. Once the granulesare fully formed they make their way via gland cell

necks which pass through the basement membrane toopen either into an epidermal cell (Figure 13) or topass between epidermal cells. The granules then maketheir way to the apical surface to be secreted to the

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Figure 7. Golgi complex (gc) in a secretory cell located below the basement membrane and muscle layers. Type II secretions are being buddedoff (arrows) as vesicles from the complex. Nucleus (N); Mitochondrion (m). (Scale = 0.25 �m)Figure 8. TEM of Type II secretory cell, which has an irregularly shaped nucleus (N) surrounded by Type II secretions (arrow), and cisternaeof granular endoplasmic reticulum (ger). (Scale = 0.5 �m)Figure 9. TEM showing the aggregation of Type II secretion vesicles in ‘packets’ following their release from the Golgi complex. (Scale = 0.3�m)Figure 10. TEM of Type II secretory cell showing the ‘packets’ of secretion. Each packet is bound by a membrane (arrows). Basement membrane(BM). (Scale = 0.5 �m)Figure 11. TEM of gland cell neck below the epidermal layer showing accumulations of Type II secretions (arrow). Epidermal rhabdoid (ER).(Scale = 0.5 �m)Figure 12. An active secretory cell located below the basement membrane producing lamellated cytoplasmic granules (Type III). The irregularshaped nucleus (N) has a prominent nucleolus (Nu). Extensive cisternae of granular endoplasmic reticulum (ger). The secretions do not appearto have an outer limiting membrane. (Scale = 0.5 �m)

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exterior (Figures 14 and 15). The granules appear tohave different banding patterns but this may be due tothe degree of maturity, the orientation of the sectionor an artefact (Figure 13). However, the majority showa distinct light/dark pattern of banding running trans-versely across the granule, with the light band 0.5 �mand the dark band 0.4 �m in width (Figure 14).

Type IVType IV secretion is contained in an irregularly shapedgland and has a dense,granular appearance (Figure 16).The origin of this secretion is unknown,but it is thoughtto be produced in the parenchyma due to the presenceof microtubules within the gland neck and the presenceof septate desmosomes joining the gland neck to thesurrounding epidermal cells.

Acid phosphatase staining at the electron micro-scope level was associated with all the secretions men-tioned above and is illustrated by the lamellated cyto-plasmic granules. Acid phosphatase staining occurredthroughout the cell, especially in the GER (Figure 17),and was present in the ducts of the gland cell neck andin between the secretions, but there was no activity onthe secretions themselves (Figure 18). It could be seento be released at the surface along with the secretorygranules (Figure 19).

Histochemical reactions were carried out at thelight microscope level using a number of stains. Type Iand Type IV secretions were not visible by lightmicroscopy. It was difficult to distinguish between theround ‘packaged’ secretions (Type II) and the lamel-lated cytoplasmic granules (Type III). However, it wasapparent that the lamellated granules gave a positiveresult with Chromotrope 2R and with eosin (when thehaematoxylin and eosin method was used), but a nega-tive result with the periodic acid-Schiff reaction, tolu-idine blue (metachromasia was absent), Alcian blueand for acid phosphatase activity. The results of thecontrols carried out for periodic acid-Schiff reaction,Alcian blue and acid phosphatase were negative, con-firming the results mentioned above. Other than thelamellated granules, the only other secretions whichwere visible were the epidermal and adenal rhabdites.

Discussion

The epidermis is important for turbellarians in that it‘serves not only as a covering epithelium, lining and

defending the body, but accomplishes a great num-ber of other important functions (locomotion, respi-ration, excretion, secretion, defensive function withthe aid of rhabdites, perception by way of nerve end-ings and sensory cells, etc.)’ (Torok & Rohlich, 1959).The structure of the turbellarian epidermis has beendescribed for a number of species (see reviews by Bedi-ni & Papi, 1974; Reisinger & Kelbetz, 1964; Riegeret al., 1991; Smith et al., 1982; Tyler, 1984; Curtiset al., 1983; Bowen & Ryder, 1974; Pedersen, 1963;Torok & Rohlich, 1959; Rieger, 1981). A multitude ofgland types has been observed in those species stud-ied, but only a few have been characterized by electronmicroscopy (Køie & Bresciani, 1973; Bowen & Ryder,1974; Bautz, 1977). Four types of turbellarian epi-dermis have been described (see Rieger, 1981; Tyler,1984): the catenulid type, the acoel-nemertodermatidtype, the macrostomatid-haplopharyngid type and theneoophoran-polyclad type. The neoophoran epidermisis characteristic for all the neoophoran orders, i.e.Lecithoepitheliata, Prolecithophora, Proseriata, Tri-cladida and Rhabdocoela and for the Polycladida. AsA. triangulata belongs to the Tricladida it falls intothis category. Unlike the epidermis of Gyratrix her-maphroditus, the epidermis of neither A. triangulatanor Bipalium adventitium (Curtis et al., 1983) becomessyncytial during its development, and thus a syncy-tial epidermis cannot be regarded as a general rule forthis group of animals. The neoophoran type of epi-dermis represents a trend towards a domination of thebasement membrane system for mechanical support(Rieger, 1981). The basement membrane of A. triangu-lata is similar to that described for Dendrocoelum lac-teum (Bedini & Papi, 1974) and B. adventitium (Curtiset al., 1983) and it is likely to play a dual role allow-ing interaction between the epidermal cells and musclecells in basement membrane formation (Hori, 1979).The epidermis of various species of land planariansmay show variability in the degree of insunken nuclei(Von Graff, 1899; Ehlers, 1985). Thus, in some pla-narians the nuclei of all the cells can be found beneaththe basement membrane, in others several are locatedbeneath the basement membrane and in yet other landplanarians like B. adventitium, most of the nuclei areinsunken around the creeping sole and in the head area(Curtis et al., 1983). As A. triangulata does not pos-sess a creeping sole, the nuclei are in the conventionalpositions, mainly above the basement membrane withsome occurring below it. No variation was observedin the present investigation between the anterior and

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Figure 13. TEM showing aggregations of Type III secretions just below the basement membrane (BM). A secretion (arrowed) can be seenpassing through the basement membrane to enter the epidermal layer. (Scale = 0.5 �m)Figure 14. TEM showing Type III secretions within a gland cell neck (large arrow) in the epidermal layer. The gland cell neck is joined to thesurrounding epidermal cells by septate desmosomes (small arrow). Adenal rhabdite (AR). (Scale = 0.5 �m)Figure 15. TEM showing the release of Type III secretions from the apical surface of the epidermis. (Scale = 0.25 �m)Figure 16. TEM of type IV secretion at the apical surface of the epidermis. The vesicles are contained within a gland cell neck which is joinedto the surrounding epidermal cells by septate desmosomes (large arrow). Note the presence of microtubules (small arrow) in the cell neck.(Scale = 0.5 �m)Figure 17. TEM showing acid phosphatase activity associated with the granular endoplasmic reticulum (arrow) in an epidermal cell.(Scale = 0.14 �m)Figure 18. Acid phosphatase activity in gland cell necks in the epidermal layer. It can be seen lying between the Type III secretory granuleswithin the duct but it is not present in the granules themselves. (Scale = 0.25 �m)Figure 19. Acid phosphatase activity associated with a secretory granule being released to the exterior (arrow). (Scale = 0.5 �m)

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posterior ends concerning insunken nuclei as there iswith B. adventitium.

Cilia are present in A. triangulata and havebeen described by SEM for other flatworms, includ-ing Microstomum lineare, Bothriomolus balticus,Archilopsis unipunctata (Reuter, 1978) and Dugesiatigrina (Smales & Blankespoor, 1978). In general, theepidermis of turbellarians is ciliated and it is the cil-ia which provide the means of locomotion for theseorganisms. While the epidermis is entirely ciliated, inmany species specialisation in terms of reduction ofcilia is common, especially the reduction or absence ofthe dorsal cilia (Williams, 1982; Rohde, 1987).

The epidermal cells of A. triangulata containsmall irregular Type I secretory granules and this isthe only secretion to be produced by the epidermalcells themselves (other than the epidermal rhabdoids:McGee et al., 1996). Release to the exterior has notbeen demonstrated. This type of secretion has beendescribed as cyanophilic by Pedersen (1963) and canbe seen in all turbellarian species possessing a cellularepidermis. The functions of the secretion may includelubrication and production of mucus, and the vesiclesmay aid in the strengthening of the epidermal layer dueto their numbers and positioning in the epidermis. Sim-ilar vesicles have been described in D. lugubris (Torok& Rohlich, 1959) and Polycelis nigra (Skaer, 1961).

According to Skaer (1961), the type I secretionexhibits a weak protein or amino acid reaction but hasa main polysaccharide component. Pedersen (1963),however, places it under the term cyanophilic althoughthe secretion may contain neutral, slightly acidic oracidic mucopolysaccharides, or mixtures of each. Itis difficult to distinguish differences in staining at thelight microscopic level and this may be due to differentcell types or different stages of elaboration (Pedersen,1963; Skaer 1961; Jennings 1957). Variation may alsoexist between the same and different species and hencedifferences may be due to an artefact. However, allsecretory cells of this type have a highly developedgranular endoplasmic reticulum.

The second type of secretion (Type II) takes theform of round, lucent vesicles which are produced bycells in the parenchyma and move to the epidermallayer via gland cell necks. When viewed under TEM,the secretions appear as if they are empty ‘packets’ butin fact they may appear homogeneous depending on theplane of section. This type of secretion has not beenfound in any other turbellarian and therefore might benovel to A. triangulata.

The third type of secretion which A. triangula-ta possesses is the lamellated cytoplasmic granule(Type III). Such granules have been recorded for D. tig-rina and P. vitta (Pedersen, 1963), D. lugubris (Torok& Rohlich, 1959, 1960), P. nigra (Skaer, 1961),D. lacteum (Gelei, 1912 in Torok & Rohlich, 1959),B. adventitium (Curtis et al., 1983) and Planaria alpina(Klima, 1961; Klug, 1960). The Type III secretory cellsin A. triangulata have all the attributes of very activesecretory cells in that they contain an extensive andwell-organised GER, multiple Golgi complexes andnumerous mitochondria. When formed, the granulesaggregate in a gland, are transported via gland cellnecks into the epidermis and the contents are pouredout onto the surface of the flatworm. Later in thesecretory cycle the endoplasmic reticulum and ribo-somes seem to disappear leaving a virtually inactivecell (Pedersen, 1963; Bedini & Papi, 1974). The situ-ation is similar in A. triangulata. The secretions havebeen termed ‘eosinophilous’ by Curtis et al. (1983)and have also been called ‘sticky’ glands as they havebeen located in the adhesive zone and are thought tobe of a mucoid nature and used for adhesion (Torok& Rohlich, 1959). Their predominantly proteinaceousnature and large content of arginine are responsiblefor most of the acidophilia (Pedersen, 1963), and theyappear to have a substance more stable and compactthan the previous secretions mentioned. This may bethe reason why they retain their characteristic shapeand structure in the epidermis. In addition, whatev-er their numbers when formed, they always retaintheir shape and individuality in the gland ducts. Fromthe histochemical staining the results agree with thoseobtained by Pedersen (1963). In particular, the positiveresult with Chromotrope 2R demonstrates the presenceof basic proteins, the positive reaction with eosin indi-cates acidophilia and the negative reaction with peri-odic acid-Schiff, toluidine blue (metachromasia) andAlcian blue shows that the granules are negative to testsfor acid mucopolysaccharides. They are also negativefor acid phosphatase activity.

There is no difference in the distribution of thissecretion over the dorsal and ventral surfaces of A. tri-angulata, as in P. vitta and D. tigrina (Pedersen, 1963).The secretions are very regular in appearance and theirstriated appearance reflects a laminar organization.From their investigation of these granules, Torok andRohlich (1960) concluded that: ‘On the basis of elec-tron microscopic and polarisation optical observationsit is assumed that filamentous micelles of the proteinmaterial of granules are oriented along the longitudi-

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nal axis of the granule. Cross-striation of the gran-ules may be explained with the presence of osmophilicgroups periodically recurring on the protein micellesor of some osmiophilic substance built in periodicallybetween the micelles’. The lamellated secretions maketheir way via gland cell necks to the apical surface ofthe epidermis prior to their release. Once they reachthe apical surface, the gland cell neck joins via sep-tate desmosomes to the apical surface of the epidermisand then, due to pressure and the gland being opened atone end, the contents are poured onto the surface of theflatworm. All the ducts are lined by regularly-spacedmicrotubules which may also assist in the transport ofthe secretory material (Bowen & Ryder, 1973).

The fourth type of secretion (Type IV) describedfor A. triangulata may be a mucoid type of secretionproduced by an insunken cell due to the presence ofmicrotubules in a presumed gland cell neck and sep-tate desmosomes binding the neck to the surroundingepidermal cells. This secretion has not been describedfor any other turbellarian and therefore it also may beconsidered novel to A. triangulata.

Acid phosphatase activity is present in associa-tion with the GER in epidermal cells but no activitywas seen on any of the epidermal secretions produced.However, as it was present along the inner lining of theducts and between the secretions, it would be releasedto the exterior along with the secretions. Those glandswhich open onto the ventral surface most likely secretea mucoid slime for locomotion (Reisinger, 1923a inReiger et al., 1991). The glands may come into playwhen the flatworms are either moving or at rest andtheir role can be seen, especially when a flatwormretracts from a stimulus. The posterior end of the bodyis firmly attached and the rest of the body is drawnback very quickly. Tyler (1976) has investigated thisbehaviour and has suggested a ‘duo-gland adhesivesystem’, that of attachment and of releasing the glandwhich may be due to the release of gland secretionsand hence may have a chemical function. It is possi-ble that two gland secretions come together to form anadhesive secretion or perhaps one alone is capable. Thesecretions may also have a role to play in the immo-bilisation of an earthworm and in the digestion of itscuticle during feeding. As yet no definite conclusionhas been reached.

There have been secretions recorded for otherturbellarians but they have not been observed in theepidermis of A. triangulata. They have been termed‘cyanophilous’ glands (Torok & Rohlich, 1959) andcan be found singly or in small groups anywhere in the

sub-epidermal regions. They consist of round gran-ules of moderate electron density and, in mature cells,the granules are packed very closely together, withorganelles rarely being seen. In other cells, which prob-ably represent early stages of the secretion cycle, anabundant granular endoplasmic reticulum is found. InD. tigrina, the granules are 0.5–0.8�m in diameter andare usually round (Pedersen,1963), whilst in P. vitta thegranules are 0.2–0.3 �m in diameter and are of mod-erate electron density (Pedersen, 1963). The secretiongranules are discharged to the outside through the epi-dermis. Long extensions of the gland cells penetratethe basement membrane and either pass between theepidermal cells or penetrate them directly (Pedersen,1963). The granules are always contained within thecell membrane from the corresponding gland cell.

In conclusion, A. triangulata possesses six typesof secretion in its epidermis, four of which have beendescribed in this paper. Epidermal rhabdoids and ade-nal rhabdites have been described in a separate paper(McGee et al., 1996). Some of the secretions arepresent in other turbellarians, for example, Type I andthe lamellated cytoplasmic granules (Type III). Othersecretions, such as Type II and Type IV, have beendemonstrated only in A. triangulata to date and con-sequently can be considered novel to this species. Onthe other hand, there are secretions present in someturbellarians which have not been observed in A. tri-angulata. That A. triangulata possesses a variety ofepidermal secretions is typical of turbellarians. Thosein A. triangulata are likely to have a number of rolesbut all will contribute to the make-up of the mucus andmay be involved in its lubricative, protective, repellent,possible neurotoxic and microenvironment-regulatingproperties. The complete elucidation of the chemicalnature and function of the individual secretions remainsan intriguing challenge for flatworm biologists.

Acknowledgments

This investigation was supported by a Postgradu-ate Training Award from the European Social Fund(to C. McGee). The authors would like to thankMr G. W. McCartney for expert photographic assis-tance.

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References

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