Cell Tissue Res. 199, 197 202 (1979) Cell and Tissue Research �9 by Springer-Verlag 1979

The Pigmentary System of Planaria

I. Morphology

Guido Palladini*, Lodovico Medolago-Albani*, Vito Margotta**, Alberto Conforti***, Antonio Carolei****

* Istituto di Biologia Generale I (Facoltd di Medicina) ** Istituto di Anatomia Comparata (Facolt& di Scienze) *** Istituto di Patologia Generale (Facoltfi di Medicina) **** I Clinica Neurologica (Facolt& di Medicina) University of Rome, Italy

Summary. The pigmentary system of the planaria, Dugesia gonocephala s.l. (Platyhelminthes, Turbellaria, Tricladida), has been studied by light and electron microscopy. The system consists of granules contained in chromatophore-l ike cells embedded in the parenchyma. The cell processes penetrate between the muscle layers and extend to the sub-epidermal basal lamina. The nature of the pigment and the comparat ive anatomical significance of the chromatophore structure is discussed.

Key words: Pigmentary system - Planaria.

The metazoan pigmentary system is of great evolutionary significance in the defence against physical environmental agents (radiations) and animal predators. It involves several cytological and histological adaptations (pigment cells, pigment granules and contractile structures such as microtubules and microfilaments) and a complex neurohormonal control system.

From the comparat ive anatomy standpoint, the data contained in the literature on lower metazoans are scanty and anything but recent, especially so far as the pigmentary system of the Turbellaria is concerned (Cloudsley-Thompson, 1945; Bagnara and Hadley, 1973). This is surprising in view of their interesting phylogenetic position and published reports which show they have a pigmentary system that has the capacity to react to light (Cloudsley-Thompson, 1945). The present investigation is one of a series designed to improve our understanding of the structure and regulatory control of this system in platyhelminths.

Send offprint requests to: Prof. Guido Palladini, Istituto di Biologia Generale I (Facolt& di Medicina), Policlinico Umberto I, 1-00100 Roma, Italy

The authors wish to thank Prof. Beniamino Guidetti, Istituto di Neurochirurgia, University of Rome, for generous loan of freeze-drying equipment and Prof. Alessandro Polleri, Istituto Scientifico di Medicina lnterna, University of Genoa, for critically reviewing the manuscript

0302-766X/79/0199/0197/$01.20

198 G. Palladini et al.

Fig. 1. Surface view of a normal live planaria showing the parallel arrangement of pigment granules along the muscle fibres

Fig. 2. Cross section through normal planaria showing the structure of a pigment cell: a deep-lying nucleus (N) and processes containing pigment granules (arrows) can be seen. E epidermis; PC parenchymal cell; M muscle fibre. H.E. staining

Materials and Methods

Two different populations of Dugesia gonocephala s.l. (Platyhelmintes, Turbellaria, Tricladida) were used: a dark-coloured one originally gathered at Rovine Canale Monterano in the Mignone River, and a light-coloured one, originally gathered at Fosso Tre Fontane.

The animals, arranged in uniform batches according to colouration and size, were kept in dechlorinated tap water at a temperature thermostatically maintained at 18-29 ~ C, in almost complete darkness. A total of 80 planaries were used.

For light microscopy the animals were preserved by freeze-drying (Edwards-Pearse rood. EPD3 Tissue Dryer), or were fixed in an extended position in Bouin's fluid. For electron microscopy, the animals were fixed in 2.5% gluteraldehyde in 0.1 M phosphate buffer, pH 7.3 at 4~ with or without post-fixation in 1.33% osmium tetroxide solution in the same buffer (Gremigni and Domenici, 1974). The paraffin-embedded specimens were sectioned in the frontal, transverse and longitudinal planes and stained with hematoxylin-eosin, or with Nissl, Azan-Mallory or Fontana-Tribondeau stains. Some sections were treated by Schmorl's histochemical method (Lison, 1960; Ganter and Joll6s, 1969-1970). Unstained sections were also examined before and after exposure to oxidizing agents. For electron microscopy the specimens were impregnated with Epon 812 and examined using a Philips EM300 apparatus, both before and after staining with lead hydroxide and uranyl acetate, and also after treatment with oxidizing agents (H20 2 10% + NH 3 20%).

Fig. 3. Schematic view of the pigmentary system in planaria: E epidermis, BM basal lamina, M F muscle fibre, PC parenchymal cell

]Fig. 4. Cross section of normal planaria showing the sub-epidermal distribution of the pigment granules. The granules are strongly argentaffinic. Fontana-Tribondeau staining

200 G. Palladini et al.

Pigmentary System of Planaria: Morphology-I 201

Results and Discussion

In the living animals (Fig. 1), and even more clearly in the fixed and stained preparations (Fig. 2), dark-brown pigment granules contained in chromatophore- like cells are seen scattered throughout the parenchyma. The cell body is triangular in cross section and the nucleus is surrounded by a few pigment granules. The cytoplasmic processes extend from the cell body to the sub-epidermal basal lamina, passing through the longitudinal and circular muscles (Fig. 3). Most of pigment granules are contained in these processes.

The pigmented cells located in the dorsal region of the animal contain small granules (0.15-0.40 Ixm), but those in the ventral region are larger and also contain signifcantly larger granules (2.9-3.2 ~tm). The granules are intensely argentaffinic to the Fontana-Tribondeau reaction (Fig. 4), and are Schmorl-positive. They become decoloured when exposed to oxidizing agents. Unlike the vertebrates, no unpigmented argentaffinic granules (premelanosome-like) have ever been obser- ved. The pigmented cells have an electron-dense cytoplasm with numerous ribosomes, round mitochondria and rough endoplasmic reticulum in the form of elongated cisternae. In the cell processes, small bundles of microtubules and microfilaments run parallel to the longitudinal axis of the extension (Fig. 5). Most of the pigment granules have a homogenous electron density and have a conspicuous limiting membrane. Some of the granules in the more ventrally located pigment cells are less electron dense, and have a characteristic "target" pattern (Fig. 6) consisting of concentric layers of alternating high and low electron density. This cell type can not be identified under the light microscope.

The pigment granules have an intrinsic electron density even without osmium post-fixation, and this is unaffected by oxidizing agents. In contrast with previous assumptions (Hyman, 1951; De Beauchamp, 1961), the present results demonstrate that the pigment cells of planarians can be considered as true chromatophores since they display a specific cell differentiation (microtubules and microfilaments) and a specific physiological activity (Cloudsley- Thompson, 1945). The planaria chromatophores, which have relatively few contractile microfilaments, anatomically resemble those described in the oligo- chaetes (Valembois, 1967), and are more primitive than crustacean chromato- phores, in which such contractile organelles are particularly well developed (Elofson and Hallberg, 1973; Robison and Charlton, 1973). Such observations are in close agreement with the phylogenetic position of the Turbellaria.

Histochemically, the planaria pigment granules may be considered to be of the melanin type (Ganter and Jollrs, 1969-1970), even though biochemical identifi- cation is lacking. Of special interest is the persistence of electron density after oxidation, which distinguishes them from ommochromes (Ganter and Jollrs, 1969- 1970), and confirms previous biochemical observations (Pinamonti, 1973).

Fig. 5. Electron micrograph of planaria pigment cell. Note pigment granules of varying sizes and the presence of tubules and filaments (arrow)

Fig. 6. Electron micrograph of planaria pigment cells with "target" pigment granules. Inset: high magnification of "target" pigment granule

202 G. Palladini et al.

I t is s ignif icant that , unl ike the ver tebrates , c o m p o u n d granules or ma tu r a t i ng forms (ver tebrate p remelanosome- l ike ) have never been observed, even though the " ta rge t granules" somewha t resemble the n o n - m a t u r e granules o f ver tebra te c h r o m a t o p h o r e s (Drochmans , 1963; Zel ickson, 1967). I t is poss ible that , in keeping with observa t ions on p l ana r i a ep ide rma l cells (Skaer, 1965), these " ta rge t -granule" con ta in ing cells m a y by in te rpre ted as s tamina l elements.

References

Bagnara, J.T., Hadley, M.E.: Chromatophores and color change; the comparative physiology of animal pigmentation. New Jersey: Prentice-Hall Inc., Englewood Cliffs 1973

Cloudsley-Thompson, J.L.: Pigments and chromatophore reactions of Polycelis nigra and Planaria lugubris. Nature 156, 294-295 (1945)

De Beauchamp, P.: Classe des Turbellari6s. In: Trait6 de Zoologic (P.P. Grass6, ed.). Tome IV/I, pp. 35- 212. Paris: Masson et C. 1961

Drochmans, P.: Melanin granules: their fine structure, formation, and degradation in normal and pathological tissues. Int. Rev. Exp. Path. 2, 357-422 (1963)

Elofsson, R., Hallberg, E.: Correlation of ultrastructure and chemical composition of crustacean chromatophore pigment. J. Ultrastruct. Res. 44, 421-429 (1973)

Ganter, P., Joll6s, G.: Histochimie normale et pathologique. Paris: Gauthier-Villars 1969-1970 Gremigni, V., Domenici, L.: Electron microscopical and cytochemical study of vitelline cells in the fresh

water Triclad Dugesia lugubris s.l.I. Origin and morphogenesis of cocoon-shell globules. Cell Tissue Res. 150, 261-270 (1974)

Hyman, L.H.: The Invertebrates - Platyhelminthes and Rhynchocoela. Vol. 2, pp. 52-434. New York: McGraw-Hill 1951

Lison, L.: Histochimie et cytochimie animales. Paris: Gauthier-Villars 1960 Pinamonti, S.: Investigations of tryptophan metabolism via kynurenine in Dugesia gonocephala

(Turbellaria, Tricladida, Paludicola). Boil. Zool. 40, 213-215 (1973) Robison, W.G. Jr., Charlton, J.S.: Microtubules, microfilaments, and pigment movement in the

chromatophores of Palaemonetes vulgaris (Crustacea). J. Exp. Zool. 186, 279-304 (1973) Skaer, R.J.: The origin and continuous replacement of epidermal cells in the planarian Polycelis tenuis

(Iijima). J. Embryol. Exp. Morph. 13, 129-139 (1965) Valembois, P.: Infrastructure des cellules pigmentaires des Lombriciens et 6tude de leur r61e dans les

h6t6rogreffes de paroi du corps. C.R. Acad. Sc., Paris, S~rie D, 265, 362-364 (1967) Zelickson, A.S.: Melanocyte, melanin granule, and Langerhans cell. In: Ultrastructure of normal and

abnormal skin. (A.S. Zelickson ed.), pp. 163-182. Philadelphia: Lea & Febiger 1967

Accepted February 17, 1979