J. Cell Set. a, 341-348 (1967) 341 Printed in Great Britain THE FINE STRUCTURE OF THE EYE OF THE LEECH, HELOBDELLA STAGNALIS A. W. CLARK* Department of Anatomy, University of Wisconsin, Madison, Wisconsin, U.S.A. SUMMARY The eye of the rhynchobdellid leech, Helobdella stagnalis, has been examined with the electron microscope. The eye is composed of a cup of pigment cells surrounding a compact mass of photo- receptor cells. In addition to pigment granules, the pigment-cell cytoplasm is characterized by mitochondria, a Golgi complex, and profiles of rough-surfaced endoplasmic reticulum. The photoreceptor cell contains a microvillous rhabdomere. The microvilli arise from the membrane of a large intracellular vesicle and obliterate much of its lumen. No connexion between the lumen of the intracellular vesicle and the extracellular space has been observed. The plasmalemma of the photoreceptor cell is folded to form thin pleats of cytoplasm which separate adjacent receptor cells from each other. No glial-like cells have been seen in the receptor cell mass. Directly sub- jacent to the microvilli and surrounding the intracellular vesicle is a tortuous and predominantly smooth-surfaced endoplasmic reticulum. A pair of centrioles is found near the rhabdomere. The cytoplasm around the nucleus is characterized by smooth- and rough-surfaced elements of endoplasmic reticulum, many mitochondria, and a Golgi complex. Proximally, the receptor cell narrows to form a nerve fibre which joins those from other cells to form the optic nerve. INTRODUCTION Leech photoreceptor cells aroused the interest of many classical cytologists and among a few of them (Whitman, 1899; Apathy, 1899) provoked a bitter and vitupera- tive controversy about the structure and derivation of this cell type. Hachlov (1910) was the last investigator of the classical period to deal with leech visual cells and, along with fresh observations of his own, he presented a comprehensive and critical survey of the literature. Of the three electron-microscopic investigations of leech eye structure, that of Hansen (1962) is the most comprehensive and encompasses the visual cell structure of seven species. Rohlich & Torok (1964) and Yanase, Fujimoto & Nishimura (1964) have restricted their investigations to the photoreceptor cells of a single arhynchob- dellid leech, Hirudo medicinalis. The paper of Rohlich & Torok presents a detailed analysis of cell fine structure. In the following investigation the fine structure of the photoreceptor cell of the rhynchobdellid leech, Helobdella stagnalis, will be described and its significance discussed. • Present address: Department of Zoology, University of California, Berkeley, California, U.S.A. Cell Sci. 2 342 A. W. Clark MATERIALS AND METHODS Two species of rhynchobdellid leeches, Helobdella stagnate, and Placobdella rugosa, were used in this study. Leeches were fixed at room temperature in veronal- buffered i % osmium tetroxide at pH 7-7 (Palade, 1952) with the addition of o-ooi M CaCl2. After fixing for 1 h, tissues were rapidly dehydrated in a graded series of ethanols and then embedded in Epon 812 (Luft, 1961). Hardened blocks were sec- tioned on a Porter-Blum ultramicrotome using glass knives. Sections were picked up on parlodion-coated, carbon-reinforced grids, stained with lead citrate (Reynolds, 1963), and examined with an RCA EMU3E electron microscope. RESULTS The eyes of Helobdella stagnate are found at the anterior end of the animal and occur as a single pair. Each eye consists of a cone or cup of pigment cells with the wide, open end of the cone pointing toward the surface of the animal. Within the pigment cup is a tightly packed conical mass of photoreceptor cells closely conforming in its shape to the inner surface of the pigment cup (Fig. 2) but separated from the pigment cells by a more or less uniform space 0-3-0-4 fi wide (Fig. 9). The pigment cup is composed of a single layer of cells, the nuclei of which are located along the outer surface (Fig. 2). The rest of each cell is composed of processes which are filled with pigment granules and run obliquely toward the cup's inner sur- face (Fig. 2). The cytoplasm of the pigment cell contains, in addition to the pigment granules, many profiles of rough endoplasmic reticulum, a prominent Golgi complex, and numerous mitochondria (Fig. 9). Each mature pigment granule is enclosed by a single membrane and, in H. stagnate, has a uniform outer diameter of about 0-35 fi (Figs. 2, 9). Close examination of pigment granules cut in grazing section reveals that they are composed of small particles, 40-50 A in diameter (Fig. 9, inset, arrow). Occasionally, pigment granules are found in the receptor cell mass, either in pigment cell processes or else free in the cytoplasm of the receptor cells. The photoreceptor cells of H. stagnate are large (the cell body being approximately 20 /i in diameter) and may be conveniently divided into several parts (Figs. 1, 2). The apical part contains the microvillous rhabdomere (Figs. 2, 4, 6) which.in many sections appears to be connected to the nuclear part of the cell by a narrow neck of cyto- plasm (Fig. 1). Proximally, each receptor cell again narrows into a nerve fibre (Figs. 1, 8). This becomes grouped with fibres from other cells and bends over the lip of the pigment cup as the optic nerve (Figs. 2, 8). The plasmalemma of the photoreceptor cell has undergone extensive elaborations. One form of these can be seen in Figs. 1, 3, 4, and 6, where careful examination of adjacent cells reveals thin, intervening layers of cytoplasm. These pleats almost everywhere separate adjacent cells from each other and are seen to originate from the receptor cells themselves (Fig. 3). In spite of an extensive search, no glial-like cell has been detected in the photoreceptor cell mass. A second, much more extensive membrane elaboration is the microvillous rhabdo- Leech eye 343 fer^t&sm Submicrovillar retlculum Fig. i. Simplified drawing of a photoreceptor cell from the eye oi Helobdella stagnalis, giving an approximate representation of its highly variable shape. Single arrows indi- cate the skein of filaments which runs the length of each microvillus. Double arrows indicate the neurofibrillar bundle. 344 A. W. Clark mere. The microvilli arise from the membrane of a large intracellular vesicle and pro- ject into its lumen. Although the microvilli obliterate much of it, the lumen of the vesicle is filled with a granular material (Fig. 4). A large number of sections has been examined for continuity between the intracellular vesicle and the extracellular space in the receptor cells of H. stagnalis and none was found. However, in view of the large size of these cells and the complex interrelationships of their membranes, such a continuity may have escaped detection. The microvilli are cylindrical in shape, approximately o-1 fi in diameter and o-6-i-4/i in length. Running down the centre of each microvillus for its entire length is a loose skein of filaments (Figs. 4, 10). The skein itself is approximately 100-130 A in diameter, and each filament is approximately 40-50 A thick. The rest of the lumen of the microvillus is filled with a slightly granular, electron-transparent material. Very often there is a slight constriction at the base of the microvillus (Figs. 1, 4, 6). Directly below the microvilli and surrounding the intracellular vesicle is an exten- sive and highly convoluted endoplasmic reticulum. Fig. 4 shows the submicrovillar reticulum in transverse section and Fig. 5 shows it in tangential section. In both figures, the cisternae have been artifactually distended and for the most part appear to be filled with an electron-transparent fluid. Scattered patches of granular material may also be seen. The reticulum membrane is predominantly smooth-surfaced, although a few ribosomes are seen to be associated with it in Fig. 5. The cytoplasm of the nuclear part of the cell is also characterized by a well- developed endoplasmic reticulum. Fig. 7 shows a region where there are distinct areas of both smooth- and rough-surfaced endoplasmic reticulum. There are several places (arrows) where the two types of endoplasmic reticulum can be seen to inter- connect. The cytoplasm of the receptor cell has a large number of mitochondria. They are found both in the submicrovillar cytoplasm and in the nuclear part of the cell, as well as in the nerve fibre (Fig. 8). A group of mitochondria is also seen in close proximity to a pair of centrioles. This grouping of mitochondria and centrioles is always located at the apical end of the cell, between the nucleus and the rhabdomere (Figs. 3, 6, 10). As in other reported examples, each centriole is composed of nine groups of three tubules arranged in a cylinder approximately 0-2 ji in diameter and 0-4/* in length. The centriolar microtubules are embedded in a finely granular matrix. Although it is not shown in any of the accompanying figures, an electron-opaque bundle of fibrils (Fig. 7) is often seen in the vicinity of the pair of centrioles. The bundle corresponds exactly in appearance to that seen in the nerve fibre (Figs. 1, 8). The receptor cell has a well-developed Golgi complex as well as many ribosomes unassociated with membranes. Numerous longitudinal and transverse profiles of microtubules are seen throughout the cytoplasm, especially in the nerve fibre (Fig. 8). In Fig. 5 microtubules are visible running parallel to the proximal surface of the submicrovillar reticulum (arrows). The cell nucleus is large and has a prominent nucleolus (Figs. 1, 2). The eye of the rhynchobdellid leech, Placobdella rugosa, has also been examined with the electron microscope. Although the pigment granules in the pigment cup are Leech eye 345 larger and more variable, and the receptor cells are larger, the fine structure of the eye is much like that just described for H.