TtBBUE 81 CELL 1975 7 (3) 453-468 Published h.r Lorrgman Grotip Ltd. Printed in Great Britain

PAULA

NEMANIC

FINE STRUCTURE OF THE COMPOUND EYE OF PORCELLlO SCABER IN LIGHT AND DARK ADAPTION ABSTRACT. The compound eyes of the terrestrial isopod Porcellio scaber comprises about 20 ommatidia. The dioptric apparatus of each ommatidia includes a biconvex cornea] lens and a spherical crystalline cone that is secreted by two cone cells. The closed rhabdom is formed by the microvillar extensions of seven pigmented retinula cells and one apical eccentric cell. All retinular axons exit the eye in one bundle. During dark-adaption pigment granules in the retinula cells rapidly withdrew from around the rhabdom and the cell periphery, and migrated basally. Rhabdoms thickened because of movement of the microvilli, and mitochondria moved medially and basally. During light adaption these processes were reversed. Multivesicular bodies became less numerous and rough endoplasmic reticulum and ribosomes proliferated during the initial stages of light adaption.

Introduction ALTHWJGH several papers have been published describing the ultrastructure of crustaceans, few have covered the compound eyes of isopods. The effect of dark adaption on isopod eyes has been reported in Oniscus asellus (Tuurala and Lehtinen, 1966, 1967) and in &iu oc~anica (Edwards, 1969). A detailed description of the ultrastructure of an isopod eye, however, is not available. Because isopod eyes appear to differ in several aspects from those of other crustaceans, this study of the compound eye of the terrestrial isopod Porcellio scaher was undertaken.*

Materials and Methods Adult Povcellio scabev were collected locally and maintained in covered glass bowls containing the litter in which they were collected. For scanning electron microscopy -

* This description

Department of Zoology and Electron Microscope Laboratory, University of California, Berkeley, California 94720. Received 29 October 29

(SEM), the animal was decapitated directly into fixative, and rough dissection of the eyes completed here. Several fixatives were tried; the best results were obtained with 2;/, glutaraldehyde in distilled water. After fixation at room temperature for 48 hr the specimen was placed in 16”/, glycerin for 24-48 hr. It was then dehydrated in an ascending ethanol series, and left in a second change of 100% ethanol for an additional 2448 hr. At this point it was either air dried or taken through a graded series of ethanol : Freon 113 and critical point dried from Freon 13 (Cohen et al., 1968). The specimen was then mounted on aluminum discs, gold coated in a Mikros vacuum evaporator equipped with a rotary stage, and examined in a Cambridge Stereoscan Mark Ila scanning electron microscope. For transmission electron microscopy, several fixatives were tried. The most routinely successful was 2 % glutaraldehyde in 0.1 M Sorenson’s phosphate buffer at is based primarily

on the eye of

P. scaber. However, an examination of the eye of Armndillidimn vu/gore reveals it to be virtually identical to that of P. sm!~er. and so the description

applies to both.

1974. 453

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454

pH 7.4, followed by post-fixation in 1% osmium tetroxide in the same buffer; all steps were carried out at room temperature. Following dehydration, the specimen was embedded in either Epon or Spurr resin. Silver or pale gold sections were cut on a Porter-Blum MT-2 ultramicrotome with a diamond knife mounted in a Healy-Westfall holder, and stained with saturated aqueous uranyl acetate for 20-25 min, followed by 45-90 set in Reynolds’ lead citrate. The sections were examined in an RCA 3G, Zeiss 9A or Siemens I electron microscope. Semi-thick (0.5 CL)sections were cut with glass knives and treated with periodic acid Schiff’s reagents (PAS). For a study of dark adaption, 34 adult P. scaber of approximately the same size were randomly divided into three groups. The isopods were placed into identical plastic Petri dishes each containing a moist piece of filter paper. Two groups (A and B) were placed under a fluorescent light of 118 V. The third group (C) was placed in a lightproof box located in a dark room. All groups were left undisturbed for 12 hr. At this time two isopods each from groups A and C were killed and their eyes dissected directly in fixative. The two group C isopods were dissected under red light, and left to fix in the dark. Group B isopods were then placed in the dark, and group C isopods were moved under the same fluorescent light as group A (control). A sample of two isopods from each group were then taken in 15 min, 45 min, 90 min, 2 hr and 4 hr. Dark-adapted eyes were fixed in the dark; light-adapted eyes were fixed under the fluorescent light. The following chart summarizes the experimental procedure where shaded areas represent darkness, and ‘ + ’ indicates one isopod sacrificed.

The experiment was repeated on six occasions and sections from approximately 60 eyes were examined. Results Ambient conditions

The compound eyes of Porcellio scaber are ovoid structures which resemble clusters of grapes. They sit in shallow depressions above the second antennae, bordered laterally by a ridge and medially by a row of tubercles (Fig. 1). Twenty to thirty ommatidia, each capped by a 50 p cornea1 facet, comprise a mature eye. Because the ommatidia are widely spaced, each facet remains circular instead of assuming the hexagonal configuration of more compact compound eyes. Several trichoid sensilla are interspersed among the ommatidia. The dioptric apparatus of an ommatidium consists of a biconvex cornea1 lens and a spherical crystalline cone. Its presumed photoreceptoral apparatus is the rhabdom, a structure 40 p long and 20 p wide, which lies immediately below the cone and is formed from the microvillar extensions of seven typical retinula cells and one eccentric cell. The rhabdom extends basally to a layered structure which has been called the fenestrated membrane (Peabody, 1939) below which each retinula cell continues as an axon. The axons of all ommatidia exit together at the base of the eyecup, forming the optic nerve. Fig. 2 diagrams the primary features of an ommatidium. I. Cuticular lens. The cuticle making up the unspecialized lens is differentiated into epicuticle and procuticle. The epicuticle consists of a 140 A outer layer of cuticulin and a 600 A layer of protein epicuticle. The

Regimen

I /////

Dark /Y//Y Time (Hours)

r+ ++ /L 12 .25 .5

++

++ .75

13

13.5

+t

++ 14

15

16

COMPOUND

EYE OF PORCELLlO

epicuticie in SEM micrographs appears as a thin coating on the lens surface (Fig. 3). The procuticle comprises a I p layer of heavily calcified exocuticle overlying the several endocuticular lamellae (Figs. 3, 4). There are I5 or more endocuticular lamellae, but most of the lens is occupied by the middle lamellae, which appear to be expanded (Fig. 4). When a cross-section of the lens is viewed, one sees concentric rings which are the curved endocuticular lamellae, similar to those found in the cornea of the xiphiuran Limuhs polyp//emus(Fahrenbach, 1968). Pore canals, I p long and 0.26 ILwide, are evident between the ommatidia, but are sparse in or absent from the lens. The epidermal cells presumed to secrete the lens are referred to as corneagenous cells. I have been able routinely to find only one corneagenous cell in an ommatidium. The cytoplasmic contents of a corneagenous cell are variable; both the distribution and quantity of organelles and inclusions depend on the molt condition of the animal. I have often found that corneagenous cells of animals between molts are either in a state of dissolution, or possess few organelles and inclusions (Fig. 5), whereas those of animals which are ready to molt are often crowded with rough endoplasmic reticulum, Golgi bodies, glycogen and mitochondria, and are in rosettes, possession of a microvillar plasma membrane. 2. C‘rystulline cone. The crystalline cone is an intracellular sphere located immediately below the corneagenous cell (Fig. 2). It consists of two hemispheres, each secreted by a cone cell, which meet medially, with their suture parallel to the long axis of the ommatidium (Fig. 6 and inset). The cone measures 17-25 TVin diameter, and comprises particlcs which resemble alpha glycogen No membrane separates this inrosettes. clusion body from the rest of the cytoplasm. Centrally. the cone contains a very dense, amorphous core of about 20 p in diameter. Surrounding the core there is a less dense ring of about 3 p in width in which the particles are arranged either in varying irregular patterns, or in rows in which the individual particles appear fused (Fig. 5 inset). In the outermost zone, which is 7 9 p wide, there is a heavy peppering of unorganized particles. An incomplete layer of mitochondria encircles the cone. These

mitochondria differ from those of retinula cells in that they are smaller (0.45 x 0.12 I*), less electron dense, and have swollen and bulbous shapes with fractured-appearing cristae. Semi-thick sections (0.5 p) of an eye treated with PAS reagents reacted positively in both control and amylase digested material. The crystalline cone in both instances appeared pale pink. The central zone of the cone showed the least positive reaction, the peripheral zone the greatest. When the sections were counter-stained with aniline blue-black, a protein stain, the crystalline cones appeared blue in the central zone and faint pinkish blue in the peripheral zone. Electron micrographs of amylase-digested specimens showed no PAS-positive material in the mitochondrial region of the muscle layer which surrounds the eye and a faint reaction in retinula and corneagenous cells, but no diminution of granules in the crystalline cone, whereas the controls contained PAS-positive material both between the mitochondria of the muscle and in retinula and corneagenous cells. 3. Retinulu cells. Each retinula cell is shaped much like an individual segment of an orange, so that it appears triangular in cross-section, with the rhabdomere located at its apex. A rhabdomere comprises two internal rows of microvilli which meet medially (Fig. 2f). Seven of the retinula cells form most of the rhabdom, with the major part contributed by cells 2. 3, 6 and 7 (see Fig. 2b-e). Distally these four cells send out two non-contiguous phalanxes of microvilli. parallel to the long axis of the ommatidium. which flank opposite sides of the cone (Figs. 2b, 8). As the rhabdomeres of cells 4 and 5 enter the rhabdom at a more proximal level, the rhabdom assumes a kidney shape with the concavity filled by the eighth (eccentric) cell’s descending process (Figs. 2c, 7). More proximally still, the rhabdom is lobed, with the rhabdomeres of cells 2, 3, 6 and 7 being more elongated than those of cells I, 4 and 5 (Fig. 2d). Near the fenestrated membrane the rhabdom is smaller. comprising fewer microvilli. The microvilli of the individual rhabdomeres are juxtaposed centrally, forming, in cross-section view, a continuous villar ring around the descending process of the eccen-

456

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tric cell (Fig. 2f). The microvilli of one rhabdomere can rarely be distinguished from those of another where they meet, but the plane along which villi from opposing sides of an individual retinula cell meet is obvious, either because the closed tips of longitudinally sectioned microvilli can be seen, or because microvilli of different orientation are juxtaposed. Retinular microvilli arise from narrow, stalked bases (0.28 p wide), each stalk giving rise to eight to twelve 4.5 p long microvilli. In cross section the villi are oval, and measure 600x 1000 A. They appear to contain a particulate material which is concentrated centrally.

Fig. 1. SEM of compound A, Antenna. x 100.

A retinula cell nucleus is a large, ovoid structure, 16 x 6 CL,located in the apical third of the cell. It is positioned peripherally and is oriented with its long axis parallel to that of the ommatidium (Fig. 2a). There is at least one nucleolus present, and much euchromatin (Fig. 7). Tubules of smooth ER often crowd the cytoplasm of a retinula cell, sometimes occurring in irregular groupings, and at other times formingwhorled structures similar to the phaosomes reported in many nauplius eyes (Fahrenbach, 1964; Elofsson, 1966) (Fig. 9). These membranous structures are more frequently proximal to the nucleus; irregular profiles of ER tubules are more common distally. Golgi bodies also occur

eye of P. scaber.

Arrows

point

to trichoid

sensilla.

Fig. 2. (a) Drawing of a longitudinal section through an ommatidium. A, axons; C, corneagenous cell; CC, crystalline cone; P, distal pigment cell; FM, fenestrated membrane; L, lens; N, retinula cell nucleus; R, rhabdomeres; RC, retinula cells; 8A, axon of eccentric cell. (b-e) Drawings of cross sections of an ommatidium taken at descending levels. The retinula cells are numbered. (f) Drawing of cross-section of an ommatidium, showing the villar orientation. MV, microvilli; N, nucleus. Fig. 3. SEM of a longitudinal section through endocuticle; EX, exocuticle. x 3300. Fig. 4. Longitudinal exocuticle. x 5000.

section

through

a cuticular

a lens. E, epicuticle;

lens. E, epicuticle;

EN,

EN, endocuticle;

EX,

Fig. 5. Cross-section through an ommatidium at the level of the crystalline cone (CC), C, corneagenous cell; DP, distal pigment cell; R, rhabdom. x 1400. Inset: Patterned particles from middle ring of the cone. x 15,000. Fig. 6. SEM of an ommatidium with the cuticle removed. CC, crystalline cones. x 850. Inset: Crystalline cone. The arrows point to the soma of the two cells. The smaller spheres are pigment granules. S, suture of cone hemispheres. x 1300. Fig. 7. Cross-section of an ommatidium at distal end of rhabdom (R). The lighter microvilli form the eccentric cell rhabdomere. Arrows point out processes between cells. SA, eccentric cell axon; MVB, multivesicular bodies; N, retinula cell nucleus; RC, retinula cells. x 4600. Inset: Corneagenous cell process (MT) adjacent to rhabdom (R). D, desmosome. x 27,000. Fig. 8. SEM of three ommatidia with the cuticle and crystalline cone removed. Arrows point out non-contiguous rhabdomeres; the central cavity in life is filled by the eccentric cell’s axon. x 1500. Fig. 9. Membranous

whorls in a retinula

cell. x 8000.

Fig. 10. Optic nerve (0). The axons bear beaded strings of ER (arrow) pherally placed mitochondria (M). G, glial cell: P, pigmented processes eyecup. x 5800.

and perioutlining

Fig. 1 I. Cross-section of ommatidium dark-adapted for 15 min. Note non-pigmented zone around rhabdom (R), and large numbers of multivesicular bodies (arrows). M, mitochondria; P, pigment granules. x 4800.

COMI'OUND

EYE

OF PORCELLIO

457

NEMANIC

near the nuclear membrane, or interspersed among mitochondria. Retinula cells contain densely aggregated mitochondria (Fig. 7), which appear long and thin (2.5 x 0.25 CL). They are concentrated laterally near the cell’s apex and near the fenestrated membrane. Retinula cells are heavily pigmented. The pigment granules, varying from 0.6 to 0.9 p in diameter, are membrane bounded, although usually their membranes are difficult to discern. In the light-adapted eye, retinular pigment is concentrated around the rhabdom (Fig. 7), outlining each rhabdomere. In addition, a single row of pigment granules also borders the plasma membrane to a level about halfway between the fenestrated membrane and the base of the eyecup. Multivesicular bodies (MVB), inclusions frequently seen in neurons, are commonly found in retinula cells. These organelles, which vary widely in size (0.36-I .25 p), comprise several 900 A vesicles (Fig. 7). The vesicles are moderately dense, containing granular material which is densest peripherally. Although MVB occur throughout retinula cells, they are most concentrated near the rhabdom and are often seen interspersed among pigment granules. They occasionally seem to be in contact with the rhabdom. Multivesicular bodies occur within neurites of retinula cells as well. Some MVB are membrane bounded but others are not. Multilamellate bodies (MLB), structures containing parallel stacks of membranes, in addition to inclusion bodies which seem to be intermediate between MLB and MVB, also

occur in retinula cells, but they are much fewer in number than the MVB. Microtubules occur in the apical cytoplasm of retinula cells, just beneath the crystalline cone. Proximal to the fenestrated membrane, retinula cells continue as large axons (8 y in diameter) (Fig. 10). Transverse bead-like strands of ER appear, and typical retinula cell mitochondria are lined up along the perimeter parallel to the axonal long axis (Fig. IO); an occasional MVB occurs as well. Pigment granules at this level are limited to the few which line the axons. The axons narrow as they approach the base of the eyecup, measuring about 0.4 p as they exit the eye. There appears to be no encapsulation of groups of axons, either from a single ommatidium, or from adjacent ommatidia as there is in Ligiu Oceania (Edwards, 1969), or of individual eccentric cell axons as occurs in Limulus polyphemus (Fahrenbach, 1911). Rather all axons exit en masse, with the glial cells (to be discussed below) enveloping them indiscriminately. As in all arthropod ommatidia thus far studied, the seven typical retinula cells of an ommatidium are joined together by desmosomes at their points of contact just before their respective microvilli juxtapose at the central part of the rhabdom (Fig. 7 and inset). These seven desmosomes continue the length of the rhabdom, ending at the beginning of the axons. The eccentric (eighth) retinula cell is located in the apical end of the ommatidium

Fig. 12. Apical entry of eccentric cell (EC) into rhabdom (R). Arrows point to distal pigment cell processes. CC, crystalline cone; M, mitochondria; R, rhabdom; S, suture between cone hemispheres. Note that the microvilli of the eccentric cell (MV) are orthogonal to those adjacent to it. x 9000. Fig. 13. Cross-section of rhabdom distorted microvilli. x 8000.

(R) dark-adapted

for 12 hr, showing

whorled,

Fig. 14. Longitudinal section through eye light adapted for 15 min after 2 hr in the dark. Note the swollen fenestrated membrane (FM), and pigment granules and mitochondria (M) located in the axons (A). G, glial cell; 0, optic nerve; R, rhabdom; RC, retinula cells. Arrows point out RER. x 2900. Fig. 15. Rhabdom from ommatidium (LS). R, rhabdomeres. x 10,000.

light adapted

for 4 hr. Note lysed retinula cell

462

and its nucleus is on a level with the crystalline cone (Fig. 12). This cell sends a long axon down the center of the rhabdom, which appears to join with the typical retinula cell axons at the level of the fenestrated membrane. The eccentric cell enters the rhabdom between retinula cells one and two (Fig. 2b), and contributes microvilli which project laterally as it traverses the rhabdom. The microvilli are not evenly distributed, but are concentrated in areas where the eccentric cell axon contacts microvilli originating from cells 2, 3, 6 and 7 (Fig. 7). They are frequently oriented perpendicular to the villi of the rhabdomeres which surround them (Fig. 12), and often they are also less electron dense (Fig. 7). The eccentric cell soma contains many typical retinula cell mitochondria, usually concentrated near the axonal hillock (Fig. 12). There are often Golgi bodies near the nucleus and many ER cisternae, giving the eccentric cell soma an appearance similar to that of other retinula cells. Pigment granules delineate the perimeter of the soma, but rarely extend into the axon (Fig. 12). The axon of the eccentric cell contains MVB, mitochondria, smooth ER, free ribosomes as well as many microtubules. 4. Pigment cells. Distal pigment cells encircle the crystalline cone (Figs. 2a, 5). These cells separate adjacent ommatidia, shielding them from one another. As many as 20 pigment cells may surround a given crystalline cone, but they are shared by adjacent cones. The pigment granules in these cells are numerous, and fill much of the cytoplasm. Ultrastructurally, the pigment appears to be identical to that of retinula cells. It can be extracted from these cells and from the retinula cells by an extended phosphate buffer rinse following glutaraldehyde fixation, suggesting that the pigment may be an ommochrome (Butenandt et al., 1960; Yoshida, 1963, 1967). Distal pigment cell nuclei are small (about 7 x 7 CL),dense structures containing much heterochromatin. Distal pigment cells have extended processes which interdigitate with the apical ends of the retinula cells, sending fingers of pigment between adjacent retinula cells (Fig. 12). The eyecup is outlined by thin processes of chromatophore cells which contain heavy concentrations of pigment granules. The same processes underlie the epidermal cells

NEMANIC

of the integument, and they appear to form a continuous dark band beneath the cephalic cuticle. Here too, the pigment granules appear identical to those of the retinula cells. 5. Processes

lying between retinula cells.

Retinula cells are separated from one another by cytoplasmic processes originating from several cells (Fig. 7). The interdigitating processes form an extensive basket, enmeshing the retinula cells. It is these processes which form the complex layering that make up the so-called fenestrated membrane; occasionally the processes consist of little more than sheets of layered plasma membranes extending along the length of the retinula cells, which then coalesce into a mesh-work at the base of the rhabdom. In other areas, cytoplasmic organelles and inclusions including pigment granules are evident. In addition to the above mentioned processes, there are seven microtubule-bearing cylinders (1.6 TVwide) which extend between the retinula cells, positioned adjacent to the desmosomes which connect the sense cells (Fig. 7 inset). These processes most often contain microtubules, an occasional mitochondrion, and a few membranous strands, and they extend only as far as the fenestrated membrane. Microtubule-bearing processes similar to these are common in insect eyes where they have been identified by serial sections as extensions of the corneagenous cells. In some ommatidia in both P. scaber and A. vulgare, I have also found corneagenous cells where the cytoplasm appears filled with crystalline bodies, and the microtubule-bearing processes of these ommatidia have a similar appearance. Below the fenestrated membrane are cells which encapsulate the axons, called glial cells because of their juxtaposition to the retinular axons (Fig. 10). Their nuclei always occur below the fenestrated membrane, but their processes seem to form part of the sheet-like processes which lie between retinula cells. The nuclei of glial cells are similar to those of corneagenous cells; that is, they are small, irregularly shaped, but often triangular structures, filled with densely staining clumps of heterochromatin (Fig. 10). Like corneagenous cells, they are unpigmented.

(‘OMPOUND

E.xperimental

EY t: OF

illumination

fORCELL/

conditions

No changes

were observed in either cuticular lenses or crystalline cones during either dark or light adaption, but extensive changes of five different types were found in retinula, corneagenous and distal pigment cells. Dark

aduption

migration. Fifteen minutes after light-adapted animais of group B were placed in the dark, pigment granules which outlined the rhabdom in light-adapted controls (group A) had pulled back, leaving a clear zone around the rhabdom containing only RER cisternae, free ribosomes, and many small vesicles (compare Figs. 7 and 12). In addition, pigment granules bordering the plasma membranes had pulled back away from the periphery of the cells. By 45 min dark-adaption pigment granules were also withdrawn from the nuclear region; many had migrated through the fenestrated membrane into the upper parts of the axons. By I2 hr dark-adaption pigment granules outlined parts of axons located well below the fenestrated membrane. 1. Pigment

2. Cellular volume increases. There was no migration of pigment granules within the distal pigment cells with darkness, but within I5 min dark-adaption distal pigment cells were less closely apposed to crystalline cones because the cell processes which lay between the crystalline cones and pigment cells swelled and thus separated them. The size of the separation did not increase with further dark adaption. The swelling of cell processes between retinula cells also caused further separation of retinula cells from each other. The fenestrated membrane also swelled (as in Fig. 14). This volume increase was evident within 15 min of dark adaption, and increased during the first 90 min of darkness. changes. Some disruption of the orderly array of rhabdomeric microvilli occurred as early as IS min dark adaption, causing the villar membranes to form extensive whorls, which are rarely seen in control eyes. This whorling was more common in 12 hr dark-adapted eyes (Fig. 13). Microvilli of any given rhabdomere which project from opposite sides of the same retinula cell and meet medially changed their 3. Microcillar

position during dark adaption so that the angle between them increased gradually from about 30” to 180’. This migration was often evident within first 15 min. By 90 min the angle between villi was about 90 and by 4 hr, it approached 180 . Because of this microvillar movement and the corneagenous cell swelling, by 45 min dark adaption some of the retinular cytoplasm, including an occasional nucleus, was forced through the perforation in the fenestrated membrane and lay below it. 4. ~~toc~ondrja~ migrations. Retinular mitochondria also migrated both medially and basally during dark adaption. Within 15 min retinular mitochondrial aggregations had moved medially from the ceil periphery (Fig. I I). After 90 min darkness, most mitochondria lay between the rhabdom and the nucleus, whereas in the light-adapted state, most lay beside or behind the nucleus. By I2 hr dark adaption most mitochondria were either dispersed among pigment graor were located well below the nules, fenestrated membrane. Multivesicular bodies were very numerous in retinula cells of dark-adapted eyes, and were often quite large, even after only I5 min (Fig. 1 I). They seemed to increase in both number and size with longer periods of darkness. Light

adaption

migration. Return to the !ightadapted state after I2 hr of darkness (group C) occurred gradually, but after I hr in light, the pigment distribution in most retinula cells was identical to that of lightadapted cells. However, in many rhabdomeres the microvilli were badly disrupted, even after 1 h of light. In these cells, pigment granules were still absent near the rhabdomere, and the area, instead, was filled with vesicles. Developing pigment granules were present in both retinula and distal pigment cells. These were not seen in the eyes from group A. I.

Pigment

2. Cellular volume decreases. By 2 hr light adaption the processes between retinula cells were identical to those in control eye. 3. Microciliar rhabdomeric

changes.

microvilli

The angle between decreased within the

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464

first 15 min of light, and after 3 hr of light adaption the angle between opposing microvilli was the same as that of the control (in those cells where microvillar disruption did not occur). 4. Retinula cell disruption. Some retinula cells lysed after 12 hr of dark followed by a period of light (Fig. 15). 5. Mtochondrial

migration. Mitochondrial migrations commenced within 15 min of light adaption, but many groups of mitochondria still lay below the fenestrated membrane (Fig. 14). After 2 hr of light the mitochondrial distribution had returned to normal. Multivesicular bodies (MVB) had decreased in both number and size after 15 min of light, and after 1 hr of light appeared as the control in both size and distribution.

6. Changes in RER. Within 15 min of exposure to light, the parts of retinula cell axons located between the fenestrated membrane and the optic nerve were filled with RER and free ribosomes, whereas both the control and dark-adapted eyes showed much less RER and very few free ribosomes. After 3 hr of light, the amount of RER present was about equal to that of thecontrol. Ribosomes, however, still were more prevalent in eyes adapted to light for 3 hr. Discussion A. Ambient illumination 1. Cuticular lens. Absence of pore canals within lenses is a feature shared with the xiphiuran Limulus polyphemus (Fahrenbach, 1968) which aids in the use of cuticle as a lens, since pore canals could cause image distortion. A second such shared feature is the predominance of endocuticle within the lenses. Because endocuticular lamellae are regularly arrayed, they are less likely to cause image distortion than are the more irregular exocuticle lamellae. Fahrenbach (1968) reported that the swirling fibers in the lenses of Limulus polyphemus acted as a light depolarizer. Porcellio scaber, reportedly able to perceive polarized light (Birukow, 1960) shows a notable decrease in the angled layering of the fibrils of the endocuticular

lamellae of the lens as compared to those of adjacent cuticle. Although I routinely found only one corneagenous cell in each ommatidium, Grenacher (1879) reported finding two in P. scaber, and in Zdothea two such cells were also identified (Peabody, 1939). Perhaps, in P. scaber two are originally present, but one degenerates, as seems to occur in Serolis (Watase, 1899). But because the corneagenous cells are difficult to fix, it is equally possible that paired cells are present in the living animal, but that the identification of both in the fixed state is rendered difficult by fixation artifacts. 2. Crystalline cone. Although most crustaceans and most insects have four-celled cones, all isopods thus far examined have displayed crystalline cones formed by only two cells (P. scaber, Grenacher, 1879; Serolis spp., Watase, 1899; Ligia spp., Hewitt, 1907; Zdothea spp., Peabody, 1939; Oniscus asellus, Debaisieux, 1944). A layer of mitochondria surrounding the crystalline cone is also found in the cladoceran, Daphnia pulex (Rohlich and Tot%, 1965). That mitochondria are significant in the development of the cone is probable, and in A. vulgare I have found well-formed mitochondria within cone cells in very early developmental stages, although in later stages mitochondria are fragmented. Similar findings have been reported for other arthropods with crystalline cones (Waddington and Perry, 1960; Burton and Stockhammer, 1969; Horridge and Giddings, 1971). The positive results of a protein stain on cones and the amylase digestion results in this study suggest that the cones of isopods may differ from those of some other invertebrates in being composed of a proteincarbohydrate complex, rather than glycogen alone. 3. Retinula cells. Since P. scaber is known to respond to both horizontally and vertically polarized light (Birukow, 1960), but not to + 45” changes in polarization, a two-channel analyzer for polarized light must be present. A likely candidate for this is the orthogonally oriented microvilli of the eccentric cell with some or all of the microvilli of adjacent retinula cells. A similar configuration in Pam&us argus in which a small basal cell

COMPOUND

EYE OF PORCELLIO

possesses microvilli normal to those of other retinula cells (Eguchi and Waterman, 1966), and the central cells of some dipterans (Melamed and Trujillo-Cenoz, 1966) have also been suggested as candidates for the same role (see Eakin, 1972). That both retinular and pigment cell pigment in P. scaher are probably ommochrome is implied by two pieces of evidence. First, the pigment is soluble in phosphate buffer. This is a characteristic of ommochrome pigments, but not of melanin pigments (see Bowness and Wolken, 1959; Butenandt ef al., 1960; Yoshida, 1963, 1967). Second. the integumental pigment of P. .sccrher,which appears identical to retinular pigment, has been identified as xanthoman ommochrome (Needham and matin, Brunet. 1957). 3. Eccentric cell. The eccentric cell of P. scaheu is unique in that it not only has a well-developed rhabdomere, but in that its soma is apical rather than basal and that its process is descending rather than ascending. In addition, the eccentric cell of P. scaber is unique for its possession of typical retinula cell inclusion bodies and organelles. Microtubule-ladened cylinders situated between retinula cells next to the rhabdom are structures common to many arthropods. However, in those arthropods in which these have been studied, they have been identified to be processes of cone cells (Rohlich and Tiirii, 1965; Horridge, 1966; Varela and Porter. 1969; Schiff and Gervasio, 1969; Pet-relet and Baumann, 1969; Horridge, 1966; Burton and Stockhammer, 1969; White. 1967). In P. scaber and A. uulgare these are processes of corneagenous cells. The appearance of the structure is identical in all cases. Why the source of those of terrestrial isopods should differ from those of other arthropods is an enigma. The role of such processes seems to be for structural support. The sheet-like desmosomes which lie between these processes and the rhabdom may also serve to maintain the structural integrity of the rhabdom. H. E~perimenfal illumination Debaisieux (1944) found that in 0. asellus (isopoda) retinular pigment migration in adaption from dark to light was completed within 30 min, but that more time was

required for the converse. This was confirmed by Tuurala and Lehtinen (1967) who reported that partial adaption from light to dark was evident in 60 min and was completed within 120 min. 1 found continuing pigment movement in dark adaption for up to 12 hr; however, all changes after 1 hr were minimal. Migration in light adaption was evident within I5 min, and was nearly completed within an hour. The differential rate of pigmentary movement between light and dark adaption could be due to different control mechanisms. Pigment migration in both dark and light adaption in many crustaceans is controlled neuro-hormonally (see Kleinholtz, 1961, 1966). If two separate neurohormones with differing rates of activity are involved in the two types of adaption, this could explain the differential rates of pigment movement. The number of developing pigment granules seen in retinula cells adapting to light suggests some pigment granules may be destroyed in periods of darkness and replaced after return to the light-adapted state. A comparable situation is seen in Daphnin pukex (Riihlich and T&ii, 1965). Kleinholtz (1961) has reported that a volume increase in dark adaption occurred in the accessory cell processes which lie between retinula cells in ldothea (isopoda), similar to that occurring in P. scaber. 1 believe this swelling serves to separate the distal pigment cells from the cone, thus allowing more light to enter the rhabdom. Microvillar disruptions after varying periods of dark adaption have been reported in several arthropod eyes. Rhabdoms of Ligia oceanica were contorted after I4 days of total darkness (Edwards, 1969). Artcmicr saliniu lost the tubular arrangement of its rhabdoms but only after 3 months of total darkness (Eguchi, 1965). Kabuta and his co-workers (1967), however, found in seven species of arthropods that if dark-adapted eyes were fixed in glutaraldehyde the rhabdoms appeared normal, but if osmium tetroxide was the primary fixative, the rhabdoms were vesiculated. But since the fixative used by Edwards (I 969) was glutarthe contorted as was mine, aldehyde, rhabdoms found in dark-adapted isopods cannot be traced to fixation alone, and rather suggests that rhabdoms of these isopods may be more susceptible to periods

NEMANIC

without light than those of some other invertebrates. In L. oceanica 14 days of darkness were needed to obtain disrupted rhabdomeres (although no results were reported for shorter periods of darkness), whereas only a few hours often gave similar results in P. scaber. Changes in the angle between microvilli during dark and light adaption have been reported for an isopod, Oniscus asellus (Tuurala and Lehtinen, 1967) which greatly resembled those which 1 found in P. scaber. Tuurala and Lehtinen (1967) suggested that this villar angle change may have to do with polarized light detection. I disagree. Such a change of villar angle has not been reported for other arthropods exposed to polarized light for extended periods. It seems more logical that this angle change of microvilli, which causes a general shortening and thickening of the rhabdom, would increase the probability of photons striking the photopigment situated on the villar membranes and thus increase the rhabdom’s efficiency in dim light. In P. scaber, MVB appear to be larger and eyes, more numerous in dark-adapted whereas in larval mosquitoes (White, 1964), a crab (Eguchi and Waterman, 1967) and another isopod (Tuurala and Lehtinen, 1967) the converse is found. If MVB serve to transport photopigment to the rhabdom as suggested by Brammer and White (1969) for mosquito larvae, one would expect an

increase during light adaption, but if MVB carry catabolites away and are actually lysosomal in nature as suggested by Rutherford and Horridge (1965), the converse would be expected. Since both situations occur, in different organisms, these inclusions may serve in both capacities. A proliferation of rough endoplasmic reticulum and of free ribosomes similar to that found in P. scaber eyes in light adaption has also been reported in larval mosquitoes that were raised in darkness (White and Sundeen, 1967), and an increase in polyribosomes, only, in 0. asellus (Tuurala and Lehtinen, 1967). This proliferation of ER and ribosomes implies an increase in protein synthesis is taking place upon return to light (as in the developing pigment). But why this RER should be confined to the distal sections of retinular axons in P. scaber is an enigma. Acknowledgements

This work was supported in part by USPHS Grant 10292 to Richard M. Eakin and NIH Grant GM 17523 to T. E. Everhart and E. R. Lewis. I thank Larry Lawler for kindly supplying many of the isopods. I thank Richard M. Eakin for reviewing the manuscript. This study formed part of the material submitted to the University of California, Berkeley, as partial fulfillment of the requirements for the Ph.D. degree in Zoology.

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