J. Cell Sci. 4, 327-351 (1969) 327 Printed in Great Britain

THE FINE STRUCTURE OF PHOTORECEPTORS IN MORIO (PHALANGIDA)

D. J. CURTIS* Department of Zoology, University of Liverpool, England

SUMMARY Fine structural studies on the eyes of the harvestman revealed the presence of microvilli in the rhabdom. The microvilli vary in length between 1 /i and 2 fi, are about 800 A wide, and curved or straight. They derive from the plasma membranes of the four retinula cells which surround the rhabdom. Approximately cylindrical in shape, the rhabdoms are about 40 /t long by about 4-6 ji in cross-diameter. Each rhabdom is situated at the centre of a retinula, and these retinulae are packed in a hexagonal array to form the retina. Distally, rhabdom fusion occurs to form a rhabdom network. The retina lies beneath the dioptric apparatus which consists of a single lens, surmounting a glassy body composed of lentigen cells. The cytoplasmic organelles of the retinula cells include mitochondria, lysosomes, sparse elements of endoplasmic reticulum, vesicular components, prominent Golgi complexes and pigment granules which possess a laminated structure. An important feature of the retinula cell is the presence of many small vesicles, about o-i /£ in diameter, clustered beneath the rhabdom. Incubation of glutaraldehyde-fixed eyes in a Gomori medium with acetylthio- choline as substrate, coupled with inhibition of controls by 62C47, indicates the presence of a presumed acetylcholinesterase in these vesicles. Similar vesicles also occur in the proximal cytoplasm of the retinula cells. Other larger vesicles, often with a core of whorled membranes, as well as dense bodies, also show acetylthiocholine-splitting activity. This latter activity is not inhibited by 62C47 and is probably the effect of lysosomal non-specific esterase. These bodies also exhibit acid phosphatase activity when incubated in a Gomori medium with /7-glycerophosphate as substrate. The presence of acetylcholinesterase activity, as distinct from non-specific esterase, in vesicles closely associated with the rhabdom and in more proximally situated vesicles is significant. It would point to the presence of an acetylcholine/acetylcholinesterase system involved in the generation and/or propagation of the sensory impulse arising from photo- stimulation of the rhabdom.

INTRODUCTION The microvillar structure of the rhabdom has been known for about a decade. Most of the work has been concerned with insect compound eyes (e.g. Goldsmith & Philpott, 1957; Fernandez-Moran, 1958) and crustacean compound eyes (e.g. Rutherford & Horridge, 1965; Eguchi & Waterman, 1966). Less attention has been paid to the photoreceptors of , among which the compound lateral eye of Limulus has attracted most attention (Miller, 1957). Simple eyes of arachnids, particularly spiders, have been studied by Miller (1957), Trujillo- Cen6z (1965) and W. E. Edwards (unpublished observations) among others. The aim of this communication is to report the extension of fine structural knowledge of eyes to another order, the Phalangida. • Present address: Department of Physiology and Biochemistry, University of Southampton. 328 D. J. Curtis No fine structural studies of phalangid photoreceptors have been made. Indeed, light-microscopical studies are rare and confined to the last century. The principal study was that of Purcell (1894), who examined the eyes of several species of harvest- men and reported that the basic, gross structure of the eyes of the different species was very similar. The present ultrastructural work confirms the findings of Purcell on gross structure and provides information about the details below the resolving power of the light microscope.

MATERIALS AND METHODS The material used for this study was obtained from large, mature specimens of Mitopus morio (Fabricius) (Phalangida, , Oligolophinae). The eyes are situated on the dorsal surface of the , mounted back-to-back on a turret and facing laterally. This ocularium was excised to provide a suitably small tissue block for fixation. The excised ocularia were fixed for 1 h in 1% osmium tetroxide, phosphate buffered (83 ml 2-26% sodium dihydrogen phosphate +17 ml 2-52% sodium hydroxide) to pH 7-4 after Millonig (1961). Tonicity is important in fixatives for fine structural studies, especially in arachnid tissues, as shown in aranaeid eye studies (W. E. Edwards, personal communication). Millonig used glucose in his fixative, but in this investigation a range of sucrose concentrations was employed instead, and a final sucrose concentration of about 0-3 M in the fixative was found to give optimal preservation of fine structure. For histochemical purposes the eyes were fixed in 5% glutaraldehyde (Sabatini, Bensch & Barrnett, 1963) phosphate buffered (Millonig, 1961) to pH 7-4, but with no added sucrose. The eyes were fixed for 2 h at room temperature (22 °C) and then stored in buffer at 5 °C. After incubation for enzyme localization, the eyes were post-fixed in osmium tetroxide as described above, but without added sucrose. The fixed tissue was acetone dehydrated and embedded in Araldite, via propylene oxide, after Luft (1961). Sections were cut in the silver-gold range using glass knives on an LKB Ultrotome. Enhanced electron contrast was obtained by staining during dehydration with uranyl acetate and by staining the sections with lead citrate (Reynolds, 1963). The specimens were examined in an AEI EM6B electron micro- scope. Stained sections were viewed using an accelerating voltage of 60 kV. In histo- chemical work, however, no staining other than the incubation procedure was used and increased contrast was obtained by operation of the microscope at an accelerating voltage of 40 kV. Histochemical procedures. Gomori methods were used to demonstrate certain enzymes in the eye tissue, at the ultrastructural level. The acetylthiocholine/copper sulphate method of Gomori (1952) was used to demonstrate acetylcholinesterase activity. The incubation medium used was that given in Pearse (i960). This contains a buffering system involving maleic acid and sodium hydroxide, glycine and mag- nesium chloride to provide Mg2+ ions for the enzyme activity. Sodium sulphate 2 provides SO4 ~ ions, which are involved in the precipitation of the reaction product. Photoreceptors in Mitopus morio 329 2 2+ Copper sulphate provides SO4 ~ ions and Cu ions. The latter react with the product of enzymic hydrolysis of thiocholine esters to produce copper thiocholine + + (Cu —S—CH2—CH2—N (CH3)3) which is precipitated as its sulphate at the site of enzyme action. The particles of reaction product are sufficiently electron-dense to be seen in the electron microscope. This technique has been successfully applied to fine structural work before, e.g. by Cauna (i960) and by Teravainen (1967). The incubation medium consisted of a solution of 10 mg acetylthiocholine iodide in 1 ml distilled water plus 4 ml of the stock solution which consisted of: copper sulphate CuSO4. sH2O, 0-3 g; glycine, 0-375 E> magnesium chloride MgCl2. 6H2O, i-og; maleic acid, 1-75 g; sodium hydroxide 4%, 30 ml; sodium sulphate Na2SO4, 40%, hot, saturated solution, 170 ml. Incubation was for 15 min at 22°C, enzyme activity being stopped by the post- fixation in osmium tetroxide. Control material was used to differentiate specific acetylcholinesterase from non-specific esterase activity. In this the inhibitor, 62C47 (i,5-bis-(4-trimethylammoniumphenyl)pentan-3-one di-iodide), was included in the incubation medium at a concentration of io~B M. A total of four blocks were used in the cholinesterase demonstration. The number of control blocks was the same as experimentals. No uninhibited material gave negative results, although occasional positive reaction was obtained in control material. In the latter the peri-rhabdomeric vesicles showing reaction product comprised only about 1% of the total. To check the presumed lysosomal nature of organelles showing non-specific esterase activity, Gomori's (1950) lead nitrate method was used. Incubation was carried out in a 0-05 M acetate buffered solution of o-oi M sodium /?-glycerophosphate at pH 5-0 for 1 h. The medium contained 0-004 M ^^ nitrate and the reaction product was formed of insoluble deposits of lead phosphate. This reaction product is sufficiently electron-dense to be seen in the electron microscope. This Gomori technique has been applied to ultrastructural studies by many workers, including Bullivant (i960), de Man, Daems, Willighagen & van Rijesel (i960), Essner & Novikoff (1961), Holt & Hicks (1961a, b), and Ogawa, Masutani & Shinonaga (1962). Nervous tissue has been shown to produce non-enzymic staining in Gomori acid phosphatase localization (Lassek, 1947). Control material was incubated in the absence of sodium y?-glycerophosphate and this differentiated the non-enzymic from the enzymic product. This localization was carried out only once. In both enzyme localizations the originally described technique involved the conversion of the initial reaction product to its corresponding sulphide. This was a necessary step for visualization in light microscopy, but not in electron-microscopic localization as the reaction product is sufficiently electron-dense to be visualized directly. The conversion to sulphide entails danger of diffusion, leading to false localization, and was not carried out in this work.

OBSERVATIONS General ultrastructure. The retina lies beneath the dioptric apparatus, which consists of a single lens and glassy body. The latter is composed of lentigen cells and is 33° D- J- Curtis separated from the retina by a pre-retinal membrane. The most prominent structures seen in the retina are the rhabdoms. These are cylindrical masses of microvilli about 40 /i long and 4-6 /t wide. They are situated at the centres of the retinulae, which pack together in hexagonal array to form the retina. Each retinula is formed from four retinula cells as originally described by Purcell (1894). All four contribute microvilli to the rhabdom by means of cylindrical foldings of their plasma membranes. One of the retinula cells, termed the central cell, gives rise to four processes at the base of the rhabdom. One of the processes projects up the centre of the rhabdom, tapering to a point 8-10 /i from the base of the rhabdom, though sometimes a slender extension may reach as far as 14-19 /£• In cross-section this process appears roughly trefoil in shape. The other three processes of the cell pass up along the side of the rhabdom, separating the three other cells, termed peripheral cells. In cross-section the central cell processes at the side of the rhabdom appear as small profiles separating the larger peripheral cell profiles. Figure 1 shows a cross-section through several retinulae; Figs. 2-5 show cross-sections at different levels. The central process at the base of the rhabdom is shown in longitudinal section in Fig. 6. The retinula cells of each retinula are held together by desmosomes. These have an appearance sug- gestive of septate desmosomes (Fig. 7). The microvilli of the rhabdom have an internal diameter of 400-1000 A and are bound by a membrane approximately 60 A thick. When fixed in osmium tetroxide alone microvillar diameters of about 800 A are in the majority, but when fixed in glutaraldehyde 600 A diameters are most plentiful. They are slightly curved in the direction of the lens and also inwards towards the centre of the rhabdom where the ends of the microvilli from different cells abut. The microvilli derived from the central cell are fairly straight and about 2-1-2-4//. in length. They are more or less straight in one plane but tend to curve towards the lens in the other. Those of the peripheral cells are more variable in length at any one level of the rhabdom, due to the crescentic form of the peripheral cells in cross-section. Those from the middle of the cell are shortest, about 1 -3-1 -5 fi as seen in cross-section, while those from the edges of the cell, which terminate nearer the centre of the rhabdom, are about 2-2-2-4 /<• long. Close to the base of the rhabdom the microvilli are straighter and of a shorter, more constant length, about 1 -o-i -4 /t, those from the centre of the peripheral cells being shortest. The ends of the microvilli at this level abut on to the central process of the central cell. These features of the microvilli may be seen in Figs. 2-6. Above the level of the central process, the microvillar ends abut on to each other. Here they are slightly more curved and more variable in length. In the most distal part of the rhabdom, the microvilli tend to be rather longer, as the cross-diameter of the rhabdom increases and the width of retinula cell cytoplasm peripheral to the rhabdom decreases. Distally, some of the rhabdoms fuse to form a network as originally described by Purcell (1894) in Opilio parietinus. The links between the rhabdoms are formed by microvillar developments of the sensory cell plasma membranes. These are similar to the microvilli of the main bulk of the rhabdoms, but more variable in length and orientation. The linking groups of microvilli are sometimes subtended by masses of Photoreceptors in Mitopus morio 331 granular, amorphous material and they then merge into the adjacent microvillar masses of distal rhabdoms. These linking microvilli are illustrated in Fig. 8, which shows a longitudinal section of the distal ends of two adjacent rhabdoms. The granular, amorphous substance which subtends the secondary groups of microvilli is also seen above the distal ends of the rhabdoms and in some places is in continuity with the pre-retinal membrane which separates the retina from the lentigen cells. At this distal level of the rhabdom, multilamellate bodies about 0-4 ji across may be seen (Fig. 12). The lamellae of these multilamellate bodies are closely associated with the microvilli of the rhabdom. In places, the continuity of the concentric lamellae is broken and the lamella is replaced by a chain of vesicles. The swollen ends of some of the lamellae suggest that these vesicles are pinched off from them. The inter- lamellar spacing in these organelles is about 500 A. The vesicles associated with them are of similar size and similarly sized vesicles are found beneath the more proximal regions of the rhabdom and also in the proximal regions of the retinula cell between the base of the rhabdom and the nucleus. Along most of their length the rhabdoms are functionally separated from each other by large numbers of pigment granules situated in the cytoplasm of the retinula cells. These pigment granules are disks ranging in diameter between 0-4/4 and 0-8 fi, with a few granules occurring outside this range. As shown in Fig. 9, isolated pigment granules in cross-section show a flattened sac-like structure, about 0-2 /i thick, with a less electron-dense core, bordered on either side by a dense lamina separated from the sac by about 750 A. The whole is encased in an amorphous substance of maximum over-all thickness around 0-7 /t. Thus oblique sectioning of a pigment granule may result in a horse-shoe shaped electron-dense profile. The retinula cells are rich in organelles besides the pigment granules. Their nuclei, situated proximally in the cells, are ovoid in shape. Their long diameters range from 5 /t to 7 /i, the short from 3 fi to 4-5 fi. Prominent Golgi complexes are abundant in the proximal region of the retinula cell, between the base of the rhabdom and the nucleus. The Golgi lamellae range in length from 0-5 /* to 4-5 fi and are oriented parallel to the long axis of the cell. Some curved lamellae are observed. The Golgi vesicles are spherical or ovoid in shape, with diameters ranging from 250 A to 500 A. Many of the vesicles are clustered around the ends of the lamellae, which appear swollen in places, suggesting vesicles pinching off from them. Mitochondria are plentiful in the retinula cells and found at all levels. Some appear to be nearly spherical or ovoid, while others are better described as vermiform and are often quite contorted. In the mid-region of the cells, the mitochondria extend in length to as much as 1-1-1-3 JJ,, with cross-diameters of 0-25-0-45/t. The mito- chondria in the distal region are somewhat smaller. The mitochondria are packed with many cristae mitochondriales, which in conjunction with their large numbers would suggest a high energy requirement of the retinula cells. Multivesicular bodies are also plentiful in the retinula cells. They occur most frequently in the proximal region of the cell, between the base of the rhabdom and the nucleus, but are sometimes found in more distal positions. These organelles consist of spherical accumulations of small vesicles. The diameters of the vesicles 332 D. J. Curtis range from 300 A to 850 A, though most lie in the 500-600 A range. The over-all diameter of the multivesicular bodies ranges from 0-2 fi to 0-5 /i, some possessing a bounding membrane whilst others do not. This may reflect a developmental difference, the organelles maturing by the formation of a bounding membrane around the clump of small vesicles. Other organelles seen in the retinula cells include electron-dense masses of varying and bizarre shapes, usually without limiting membranes. These bizarre bodies ranging in size from 0-5/* to i-o/t, occasionally up to 2 /.(., are probably lipid in nature. The proportions and distribution of saturated and unsaturated lipids, with their differing affinities for osmium tetroxide, could be responsible for the peculiar shapes seen in these bodies. They have been observed before in various tissues and described as osmiophilic bodies, lipoidal granules, etc. (e.g. Cardell, 1962). Apart from fairly sparse elements of both rough and smooth endoplasmic reticulum other organelles present are probably lysosomal in nature. They include various bodies, dense bodies, multilamellate bodies and vesicular structures, as well as autophagic vacuoles. As described below, these organelles possess both esterase and acid phosphatase activity, which would confirm their lysosomal status. A most important feature of the retinula cells is the presence of vesicular and reticulate elements beneath the rhabdom. These are closely associated with the microvilli of the rhabdom and, as described below, possess presumed acetylcholin- esterase activity. These peri-rhabdomeric organelles usually appear as circular or elliptical profiles. In some favourable sections, however, they appear as contorted and anastomosing tubules forming a peri-rhabdomeric reticulum, as shown in Fig. 13. Serial sections show that both vesicular and tubular elements are involved. The cross-diameters range from as little as about 400 A to as large as 0-2 /6 or, rarely, as large as 0-3 /t. Vesicles in glutaraldehyde plus osmium tetroxide fixed material tend to be larger, with diameters mostly in the range 800-1000 A, than in material fixed in osmium tetroxide alone, where they lie mainly between 400 A and 600 A. There is a close association between these structures and the microvilli of the rhabdom and vesicles are sometimes seen within microvilli. On rare occasions, vesicles may be observed in the intercellular space close to the microvillar bases. The position of these peri-rhabdomeric elements relative to the rhabdom is illustrated in Figs. 14 and 15, showing transverse sections. Histochemistry. The data reported here concern the presence of a presumed acetylcholinesterase in the peri-rhabdomeric vesicles. Incubation of eyes in the Gomori choline esterase medium produced deposits of small (from 50 A to 130 A) electron-dense particles in the peri-rhabdomeric vesicles. Some reaction product was also formed in other cytoplasmic vesicles and in larger organelles. Mitochondria, multivesicular bodies and, as a rule, Golgi complexes showed no activity. The cytoplasmic vesicles, most of which are situated in the retinula cell cytoplasm proximal to the base of the rhabdom and at the level of the nucleus, show a wide range of diameters. They vary between 800 A and as much as i/i and occasional vesicles may be seen as large as about 2 ft. Many of these contain a core of whorled membranous lamellae. These show alternating dark and light bands with a periodicity of about Photoreceptors in Mitopus morio 333 20-30 A and a comparable width. The particles of reaction product are seen attached to the surface of these membranes (Fig. 22). In smaller vesicles without the lamellated core, the reaction product appears located principally around the periphery of the vesicles. Figures 16 and 18 show medium-power fields showing reaction product in the peri-rhabdomeric vesicles and they compare with control material shown in Fig. 17. The precise, peripheral location of the reaction product within these vesicles is illustrated in Fig. 19, which compares with control material at the same magnification in Fig. 20. The control material, incubated in the presence of IO~BM 62C47, shows practically no reaction product in the peri-rhabdomeric vesicles. This indicates inhibition of the enzyme. Randomly distributed particles of the reaction product, occasionally seen in the sections, are also present in control material (Fig. 17) and are thus not due to the enzyme action. More importantly, the inhibition with 62C47 differentiates between the enzymes of the peri-rhabdomeric vesicles and those of the cytoplasmic vesicles. Most of the latter are not inhibited, and those which are appear similar in size and shape to the peri-rhabdomeric vesicles. 62 C47 is a specific inhibitor of acetylcholinesterase. Pearse (i960) gives the inhibition by 62C47 of rat brain and intestine acetylcholinesterase and non-specific choline esterase as 94% and 1 % respectively at an inhibitor concentration of 5 x io"6 M, and 101% and 3% res- pectively at 2 x IO~5M. Thus the enzyme in the peri-rhabdomeric vesicles which is capable of hydrolysing acetylthiocholine and is inhibited by 62C47 may be presumed to be specific acetylcholinesterase. The esterase of the larger cytoplasmic organelles, however, is non-specific. An indication of the distribution of the latter structures is given in the low-power field shown in Fig. 21. The appearance of these cytoplasmic organelles and the presence of non-specific esterase in them would suggest that they are lysosomal in nature. This is supported by the presence of acid phosphatase activity. Incubation in a standard Gomori acid phosphatase medium shows the presence of enzymic activity in these organelles capable of hydrolysing sodium /?-glycerophosphate at pH 5-0. This is illustrated in Figs. 23 and 24, which compare with Fig. 25 showing control material incubated in the absence of /?-glycerophosphate. The proximal region of the retinula cell contains many profiles of Golgi lamellae, which appear to bud off Golgi vesicles. Some of the larger Golgi vesicles exhibit acetylcholinesterase activity. These possibly represent the site of the formation of acetylcholinesterase-positive vesicles. Other origins are possible. The vesicles associated with the distally located multilamellate bodies may be precursors, or the acetylcholinesterase positive vesicles may be produced directly from the microvilli of the rhabdom, though the latter structure shows no acetylcholinesterase activity.

DISCUSSION The results presented in this communication expand our knowledge of the arthropod photoreceptor. The presence of microvilli in the phalangid eye provides another example in support of Eakin's hypothesis on photoreceptor evolution (Eakin, 334 D. J. Curtis 1965). According to this theory the , along with other protostomes, as well as acoels and psuedocoels, have repressed the ciliary component of primitive photo- receptors (seen in, for example, Euglena) and developed the rhabdomeric photo- receptor. The arthropods, in general, utilize more or less straight lateral villi from the photosensory cell plasma membrane to form the rhabdom. This is demonstrated here in phalangids. Eakin conjectures that the other evolutionary line of photo- receptors led to the deuterostome type and utilized modifications of the primitive cilium to develop specialized photosensory structures, e.g. the outer segments of vertebrate rods and cones. The basic principle common to these photoreceptor structures is the formation of a multilamellar membrane system, packed with photo- sensitive pigment, ideally suited for the absorption of photons. The microvilli comprising the rhabdom are remarkably uniform throughout the arthropods so far studied. They vary in length from as short as about 0-35-0-5 /«. in spiders (Lycosa: Trujillo-Cenoz, 1965; Tegenaria domestica: D. J. Curtis, unpub- lished observations) to as long as the 2-4/* microvilli reported here in phalangids. The transverse diameters are more constant, usually approximately 400-500 A, though occasionally up to 1200 A. A few examples will illustrate this point. Eguchi & Waterman (1966) give in tabular form the rhabdom dimensions of ten species of crustaceans. The microvilli range in transverse diameter from 0-05 /t to o-i /.i. Many insects have been shown to have similarly sized microvilli. Thus Fernandez-Moran (1958) reports the fine structure of the eyes of Musca domestica, Drosophila melano- gaster, Apis mellifera, the tropical moth Erebus odora, 'Skipper' butterflies (Hes- periidae), Dissosteira and tropical dragonflies (Odonata). In all these species, the microvilli are 400-1200 A in diameter. The phalangid eye shows other features seen in other arthropod eyes, in addition to the microvillar rhabdom. For example, the multilamellate and multivesicular bodies have been reported in locust eyes (Horridge & Barnard, 1965) and in crustacean eyes (Rutherford & Horridge, 1965; Eguchi & Waterman, 1966). The multivesicular bodies are typical and constant elements of the apical portion of the spider photo- receptor cell (Trujillo-Cen6z, 1965). The vesicular and tubular elements beneath the rhabdom are also present in the spider eye, e.g. in Lycosa (Trujillo-Cen6z, 1965) and in Tegenaria domestica (D. J. Curtis, unpublished observations). It has been suggested that these vesicles and tubules may be involved in pinocytosis (Baccetti & Bedini, 1964). The peri-rhabdomeric vesicles shown in Fig. 16 have clear zones associated with them and appear much larger than those shown in Figs. 13-15. However, in the author's opinion the peri-rhabdomeric vesicles labelled as such in Figs. 13-15 are the same as those in Fig. 16. They appear different because of preparative procedure. Thus the vesicles in material fixed in osmium tetroxide alone have diameters mainly in the range 400-600 A. In material fixed with glutaraldehyde most have diameters in the range 800-1000 A. This contrasts with the microvilli which tend to have larger diameters (about 800 A) in osmium tetroxide fixed material than in glutaralde- hyde fixed tissue (about 600 A). The distal fusion of the rhabdoms seen in Mitopus eye, and in other phalangid Photoreceptors in Mitopus morio 335 eyes, is an unusual and perhaps unique feature. It may serve to increase the sensitivity of the eye in these principally nocturnal . The rhabdom network certainly increases the surface area presumably sensitive to incident photons. Thus in the mid-region of the retina, where there are no secondary linking groups of microvilli, the rhabdoms occupy about 30-40% of the total cross-sectional surface area. Distally, however, the presence of the secondary microvilli increases the sensory area to about 60% of the total cross-sectional area. Excitation by light of a secondary microvillar group may possibly spread to both of the rhabdoms which it links. Or, excitation may spread from one rhabdom to another via linking microvilli. Thus two retinulae might be stimulated by perhaps only a single photon, which would result in nerve impulses passing along two sensory axons instead of one. The effect of this would be an increase in sensitivity at the expense of acuity. The desmosomes which bind the retinula cells of a single retinula have, at high magnification, the appearance of septate desmosomes, showing bands of different electron density linking the adjacent plasma membranes. This type of desmosome, and others, have been shown to provide sites of low resistance to the cell-to-cell movement of diffusible substances like ions and water (Loewenstein & Kanno, 1964; Loewenstein et al. 1965; Penn, 1966). This would suggest that interaction might take place between the retinula cells of a retinula, i.e. that the unit of reception is the retinula rather than the retinula cell. In support of this, electrical interaction between retinula cells of the ommatidium of Limulus has been demonstrated (Borsellino, Fuortes & Smith, 1965). The demonstration of acetylcholinesterase activity in the photoreceptor cells is interesting. The enzyme is located in close proximity to the presumed photosensory structure, the rhabdom, as well as in the more proximal region of the retinula cells. The acetylcholine/acetylcholinesterase system is known to be involved in many nervous tissues and has been demonstrated, for example, in toad photoreceptor cells (Sabatini et al. 1963). The presence of the enzyme in vesicles so closely associated with the sensory structure suggests very strongly that the acetylcholine/acetyl- cholinesterase system is involved in some way in the transduction of photic stimulation into sensory nerve impulse. Whether the system is directly effected by photons, or is involved in some intermediate stage in the process, possibly amplifying the photo- sensory signal, requires a great deal more work involving different techniques to elucidate. Acknowledgement is due to S.R.C. for a supporting grant; to W. E. Edwards for advice on fixation; to the late Professor R. J. Pumphrey, in whose department this work was carried out, for his interest; to Drs C. L. Smith and W. J. W. Hines of the Department of Zoology, University of Liverpool, for helpful discussions; to Dr R. G. Pearson of the Department of Zoology, University of Liverpool, for valuable advice, and to Mr D. T. Stratton for technical assistance. 336 D. J. Curtis

REFERENCES BACCETTI, B. & BEDINI, C. (1964). Research on the structure and physiology of the eyes of a Lycosid spider. Arclis ital. Biol. 102, 97-122. BORSELLINO, A., FUORTES, M. G. F. & SMITH, T. E. (1965). Visual responses in Linmlus. Cold Spring Harb. Syrnp. qiiant. Biol. 30, 429-443. BULLIVANT, S. (i960). The staining of thin sections of mouse pancreas prepared by the Fern&ndez-Moran helium II freeze substitution method. J. biophys. biochem. Cytol. 8, 639-647. CARDELL, R. R. (1962). The origin of the thyroidectomy cell in the salamander. In Vth Int. Congr. Electron Microsc. vol. 2. (ed. S. S. Breese, Jr.), p. WW 3. New York and London: Academic Press. CAUNA, N. (i960). The distribution of cholinesterase in the cutaneous receptor organs, especially touch corpuscles of the human finger. J. Histocheni. Cytocheni. 8, 367-375. EAKIN, R. M. (1965). Evolution of photoreceptors. Cold Spring Harb. Symp. quant. Biol. 30, 363-37°- EGUCHI, E. & WATERMAN, T. H. (1966). Fine structure patterns in crustacean rhabdoms. In The Functional Organization of tlie Compound Eye (ed. C. G. Bernhard), pp. 105-124. London: Pergamon. ESSNER, E. & NOVIKOFF, A. B. (1961). Localization of acid phosphatase activity in hepatic lysosomes by means of electron microscopy. J. biophys. biochem. Cytol. 9, 773-784. FERNANDEZ-MORAN, H. (1958). Fine structure of the light receptors of the compound eyes of insects. Expl Cell Res. (Suppl.) 5, 586-644. GOLDSMITH, T. H. & PHILPOTT, D. E. (1957). The microstructure of the compound eyes of insects. J. biophys. biochem. Cytol. 3, 429-440. GOMORI, G. (1950). An improved histochemical technique for acid phosphatase. Stain Technol. 25, 81-85. GOMORI, G. (1952). Microscopic Histochemistry. University of Chicago Press. HOLT, S. J. & HICKS, S. M. (1961a). Studies on formalin fixation for electron microscopy and cytochemical staining purposes. _7. biophys. biochem. Cytol. 11, 31-46. HOLT, S. J. & HICKS, S. M. (19616). The localization of acid phosphatase in rat liver cells as revealed by combined cytochemical staining and electron microscopy. J. biophys. biochem. Cytol. 11, 47-66. HORRIDGE, G. A. & BARNARD, P. B. T. (1965). Movement of palisade in locust retinula cells when illuminated. Q. Jl microsc. Sci. 106, 131-135. LASSEK, A. M. (1947). The stability of so-called axonal acid phosphatase as determined by experiments in its ' stainability'. Stain Technol. 22, 133-138. LoEWENSTEiN, W. R. & KANNO, Y. (1964). Studies on an epithelial (gland) cell junction. I. Modifications of surface membrane permeability. J. Cell Biol. 22, 565-586. LOEWENSTEIN, W. R., SOCOLAR, S. J., HlGASHLNO, S., KANNO, Y. & DAVIDSON, N. (1965). Intercellular communication: renal, urinary bladder, sensory and salivary gland cells. Science, N. Y. 149, 295-298. LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. biophys. biochem. Cytol. 9, 409-414. MAN, J. C. H. DE, DAEMS, W. T., WILLIGHACEN, R. G. J. & VAN RIJESEL, T. G. (i960). Electron dense bodies in liver tissue of the mouse in relation to the activity of acid phos- phatase. J. Ultrastruct. Res. 4, 43-57. MILLER, W. H. (1957). Morphology of the ommatidia of the compound eye of Limulus. J. biophys. biochem. Cytol. 3, 421-428. MILLONIG, G. (1961). Advantages of a phosphate buffer for OsO4 solutions in fixation. J. appl. Phys. 32, 1637. OGAWA, K., MASUTANI, K. & SHINONAGA, Y. (1962). Electron histochemical demonstration of acid phosphatase in the normal rat jejunum. J. Histocheni. Cytochem. 10, 228-229. PEARSE, A. G. E. (i960). Histocliemistry: Theoretical and Applied. London: Churchill. PENN, R. D. (1966). Ionic communication between liver cells. J. Cell Biol. 29, 171-174. PURCELL, F. (1894). Uber den Bau der Phalangidenaugen. Z. wiss. Zool. 58, 1-53. Photoreceptors in Mitopus morio 337 REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy.^. Cell Biol. 17, 208-212. RUTHERFORD, D. J. & HORRIDCE, G. A. (1965). The rhabdom of the lobster eye. Q. Jl microsc. Set. 106, 119-130. SABATINI, D. D., BENSCH, K. & BARRNETT, R. J. (1963). Cytochemistry and electron micro- scopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19-58. TERAVAINEN, H. (1967). Electron microscopic localization of cholinesterase in the rat myoneural junction. Histochemie 10, 266-271. TRUJILLO-CEN6Z, O. (1965). Some aspects of the structural organization of the arthropod eye. Cold Spring Harb. Symp. quant. Biol. 30, 371-382. TRUJILLO-CEN6Z, O. & MELAMED, J. (1966). Electron microscope observations on the peripheral and intermediate retinas of dipterans. In The Functional Organization of tlie Compound Eye (ed. C. G. Bernhard), pp. 339-361. London: Pergamon.

{Received 15 May 1968)

Cell Sci. 4 338 D. J. Curtis

ABBREVIATIONS ON PLATES a amorphous substance mlb multilamellate body apv autophagic vacuole mv microvilli of rhabdom bb bizarre body mvb multivesicular body c central cell P peripheral cell cp central process P8 pigment granule d desmosome prm pre-retinal membrane db dense body r rhabdom 8 Golgi complex s secondary microvilli I linking microvilli t tubules (peri-rhabdomeric) ly lysosome V vesicles (peri-rhabdomeric) in mitochondrion

Fig. i. This low-power electron micrograph shows several retinulae in cross-section. The hexagonal mode of packing of the retinulae and the separation of the rhabdoms (r) from one another by pigment granules (pg) is well shown. The central cell (c) is visible as small profiles, most without pigment granules, between the larger peripheral cells (p), packed with pigment granules. Osmium tetroxide fixed, uranium and lead stained, x 9000. Photoreceptors in Mitopus morio 339 340 D. J. Curtis

Figs. 2-5. Low-power electron micrographs at different levels of the retinula. Osmium tetroxide fixed, uranium and lead stained, x 9000. Fig. 2. At the extreme proximal end of the rhabdom. The central cell (c) has yet only divided into two parts which, with two of the peripheral cells (p), are starting to contribute microvilli to the rhabdom (r). The third peripheral cell of this retinula is just out of the picture at the left. The rhabdom at the top left of the micrograph shows the slightly lateral origin of the central process (e/>). Some of the pigment granules are irregular in shape due to oblique sectioning. A bizarre body (bb) is visible in the central cell. Fig. 3. Slightly distal to Fig. 2, the central process now appears in the centre of the rhabdom, and the central cell (c) as three small profiles separating the peripheral cells (/>). The oval bodies in the central process are mitochondria. Fig. 4. Distal to the level of the central process the microvilli abut on to each other at the centre of the rhabdom. The central and peripheral cells are packed with pigment granules. Fig. 5. At the extreme distal end of the rhabdoms, secondary groups of microvilli (s) are subtended by amorphous substance (a) and link the rhabdoms (r) as indicated by the arrow. Photoreceptors in Mitopus morio 341 342 D. J. Curtis

Fig. 6. This electron micrograph shows, in longitudinal section, the proximal portion of a single rhabdom (r). The central process (cp) projects from the central cell (c) at the base of the rhabdom. Another process from the central cell is visible at the side of the rhabdom. One of the peripheral cells (p) is at the other side and it contains an elongated Golgi complex (g) at the extreme left of the picture. Osmium tetroxide fixed, uranium and lead stained, x 7500. Fig. 7. This high-power electron micrograph shows a junction between two retinula cells. The intercellular gap between the thickened plasma membranes is traversed by regions of higher electron density, some of which are indicated by arrows. Osmium tetroxide fixed, uranium and lead stained, x 200000 Fig. 8. Electron micrograph showing the distal ends of two adjacent rhabdoms (r). Secondary microvilli (/) are seen linking the two rhabdoms, which are underlain by many peri-rhabdomeric vesicles (v). Amorphous substance (a) is present distal to one of the rhabdoms, underneath the pre-retinal membrane (prm) which separates the retina from the lentigen cells. Osmium tetroxide fixed, uranium and lead stained, x 25000. Fig. 9. The two isolated pigment granules seen here in cross-section show the sac-like structure of these organelles. At either side of the long sac a lamella is present, surrounded by an amorphous material, slightly granular in appearance. Osmium tetroxide fixed, uranium and lead stained, x 12000. Fig. 10. Longitudinal section in the region of the proximal end of a rhabdom (r), showing the slightly lateral origin of the central process (arrow). The microvilli of the rhabdom are sectioned transversely and peri-rhabdomeric vesicles (D) are plentiful. Organelles present include mitochondria (m) and multivesicular bodies (mvb). A desmosome (d) appears as a thickening of the cell membranes. Osmium tetroxide fixed, uranium and lead stained, x 10000. Fig. 11. This electron micrograph shows some of the organelles found in the proximal part of the retinula cell. They include mitochondria (m), multivesicular bodies (mvb), Golgi apparatus (g) and dense bodies (db). The latter, which often, as here, show some internal structure, represent a type of lysosome. Osmium tetroxide fixed, uranium and lead stained, x 24000. Photoreceptors in Mitopus morio 343 344 D. J. Curtis

Fig. 12. Multilamellate bodies (mlb) are situated at the periphery of the distal rhabdom (r). They are in continuity with the rhabdomal microvilli and also with peri-rhabdomeric vesicles (v). Osmium tetroxide fixed, uranium and lead stained, x 75000. Fig. 13. Tubular elements (t) are seen here in an oblique longitudinal section of a rhabdom (r). Intermingled with this peri-rhabdomeric reticulum are peri-rhabdomeric vesicles (v). Osmium tetroxide fixed, uranium and lead stained, x 60000. Photoreceptors in Mitopus morio 345

13 346 D. J. Curtis

Fig. 14. This electron micrograph shows a cross-section of a single rhabdom (r). The positions of the peripheral (p) and central (c) cell profiles are clear. Peri- rhabdomeric vesicles (v) are present all around the periphery of the rhabdom. Osmium tetroxide fixed, uranium and lead stained. X 27000. Fig. 15. At higher magnification, the peri-rhabdomeric vesicles (i>), with associated lamellar elements, are more obvious, x 94000. Pliotoreceptors in Mitopus morio 347 348 D. J. Curtis

Fig. 16. This electron micrograph shows unstained material which has been incubated to demonstrate acetylcholinesterase. Small particles of reaction product are present in most of the peri-rhabdomeric vesicles (arrows), in which they are usually distributed peripherally. Some particles are also randomly distributed over the section, but these are also present in control material. Glutaraldehyde and osmium tetroxide fixed, no counterstaining. x 28000. Fig. 17. In control material incubated in the presence of 62C47 the peri-rhabdomeric vesicles (arrowed) show little or no reaction product. Occasional positive vesicles (double arrow) are seen. Some faint particles are randomly distributed. Glutaralde- hyde and osmium tetroxide fixed, no counterstaining. x 28000. Fig. 18. A higher-power electron micrograph of material incubated for acetylcholin- esterase shows reaction product in the peri-rhabdomeric vesicles (arrows). Glutar- aldehyde and osmium tetroxide fixed, no counterstaining. x 40000. Fig. 19. High-power electron micrograph of unstained material shows the presence of reaction product indicating acetylcholinesterase distributed around the periphery of peri-rhabdomeric vesicles (v). At upper right-hand corner a vesicle (arrowed) has been sectioned tangentially. The microvilli (mv) of the rhabdom show no reaction product. Glutaraldehyde and osmium tetroxide fixed, no counterstaining. x 75 000. Fig. 20. Control material, at the same magnification, shows the lack of reaction product in the peri-rhabdomeric vesicles (u) when incubated in the presence of 62C47. Indeed, in the absence of reaction product, the vesicles are difficult to discern. Glutaraldehyde and osmium tetroxide fixed, no counterstaining. x 75000. Photoreceptors in Mitopus morio 349 350 D. J. Curtis

Fig. 21. Low-power field in the proximal region of retinula cells, incubated for esterase with acetylthiocholine as substrate, shows reaction product in lysosomes (ly) and the distribution of these organelles. Glutaraldehyde and osmium tetroxide fixed, no counterstaining. x nooo. Fig. 22. High-power electron micrograph shows the location of esterase reaction product particles on the surface of whorled membranes forming a core to a cyto- plasmic vesicle. Glutaraldehyde and osmium tetroxide fixed, no counterstaining. x 170000. Fig. 23. Acid phosphatase is demonstrated here in an autophagic vacuole (apv) and two dense bodies (db). Randomly distributed particles of lead phosphate are present, but are not as obvious as the product in the lysosomes (compare with Fig. 25). Fig. 24. Dense deposits of acid phosphatase reaction product in dense bodies (db) contrast markedly with the random precipitate. Glutaraldehyde and osmium tetroxide fixed, no counterstaining. x 20000. Fig. 25. Control material, incubated in the absence of substrate, shows no more reaction product in the dense bodies than elsewhere in the cytoplasm. This indicates the specificity of reaction product seen in Figs. 23 and 24. Glutaraldehyde and osmium tetroxide fixed, no counterstaining. x 37000. Pliotoreceptors in Mitopus morio 351

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