Fine structure and spectral sensitivities of retinular cells ... - Springer Link

Z. vergI. Physiologic 71,201--218 (1971) 9 by Springer-Verlag 1971

Fine Structure and Spectral Sensitivities of Retinular Cells in the Dorsal Sector of Compound Eyes in the Dragonfly Aeschna EISUKE EGUCHI* Zoologisches Institut der Universit/it ~finchen** Received November 16, 1970

Summary. 1. Retinular fine structure observed with the electron microscope and receptor potentials of single retinular cells responding to equal quantum narrow band monochromatic stimuli between 327 and 615nm have been studied in the dorsal sector of the compound eye of Aeschna cyanea. 2. Each retinula comprises 8 cells: 5 distal retinular cells, 2 proximal retinular cells and ] small cell without a rhabdomere. Nevertheless, the small cell sends an axon to the lamina. In terms of microvillus directions seen in cross section the distal rhabdom has three parts with an angular difference between them of 120 ~ The proximal rhabdom lacks one of these three parts and is made up of two, again separated in microvillus direction by 120 ~ (240~ 3. Basically the dorsal area of the eye has two types of receptor cells: a UV type (~max 356 nm) and a green type (Xmax 475-519 nm with a secondary peak at 356 nm). However, other response types, most likely derived from the green cells, were fairly frequently recorded: blue type, double type (green plus blue) and shifting type (alternately green and blue). 4. Close control of stimulus direction shows that green cells giving a single peak at 475-519 nm to axial or near axial light rays, develop a second peak at 458 nm when the stimulus direction deviates more strongly from axial. 5. Comparison of structural and electrophysiological evidence suggests that the distal retinular cells are the green receptors, the proximal units the UV receptors but direct evidence is needed. Introduction Spectral sensitivities in the v e n t r a l p a r t of t h e c o m p o u n d eye of t h e dragonfly, Aeschna cyanea, h a v e been studied p r e v i o u s l y b y intracellular electrical recording ( A u t r u m a n d Kolb, 1968). Two different cell t y p es were identified one with ~max a t 519 nml the o t h er with a 2max a t 412 to 432 n m (both h a d s e c o n d a r y m a x i m a in t h e region 356-370 rim). The p r es e n t work is d e v o t e d to th e s t u d y of t h e dorsal p a r t of this same c o m p o u n d eye. I n a d d i t i o n to the electrophysiological s t u d y with * Supported by a fellowship from Alexander yon Humboldt-Stiftung, Bundesrepublik Deutschland. ** Present address: Biology Department, Yokohama City University, Mutsuurn, Kanazawa-ku, Yokohama 236, Japan.

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intracellular microelectrodes, t h e eye's fine s t r u c t u r e was observed w i t h t h e electron microscope, These d a t a thus p e r m i t t h e correlation of structure a n d function in t h e dorsal p a r t of Aeschna's eye a n d an analysis of t h e f u n c t i o n a l differences b e t w e e n its dorsal an d v e n t r a l parts.

Material and Methods The dragonflies (Aeschna cyanea Mfiller) used in this experiment were collected as nymphs in small ponds near Munich during the summers of 1967 and 1968. They were raised to imagos in the laboratory and used for electrophysiological experiments 2-5 days after emerging. The experimental methods and the instruments for eleetrophysiological investigations were the same as those described in the paper of Autrmn and Kolb (1968) except for the glass fiber light guide used in some of the present experiments to align the stimulus direction with an ommatidial axis. A xenon-lamp (900 watts) was used as a light source, and 18 different narrow wavelength bands from 336 to 650 nm were obtained by linear interference filters. The dragonfly heads were severed near the thorax, and the heads were mounted on a specimen holder with wax. The indifferent electrode was put in the thorax. The recording electrodes (filled with 3M KCI and with resistances between 35-70 megohms) were vertically inserted into the retinular layer from a small hole opened in the cornea with a sharp razor blade. Receptor potentials were picked up by a cathode-follower input stage leading into a cathode oscilloscope. The light stimulus lasted 75 or 100 msec and its intensity was controlled with neutral filters. In these experiments, only the right eyes were used. For electron microscope observations the compound eyes were severed from the head, and immersed in 5 % glutaraldehyde buffered at pH 7.4 with 0.1 M phosphate buffer solution for 1-2 hours. After washing briefly with the same buffer solution, the eye was refixed with 2 % OsO4 buffered at pH 7.4 with the same buffer solution for 2 hours. During this second fixation, the compound eyes were cut into smaller pieces with a razor blade. The specimens were dehydrated through a series of acetone solutions followed by propylene oxide and finally embedded in Epon (Durcapon). Thin sections were cut with a LKB microtome and stained by saturated uranyl acetate and by lead tartrate (Reynolds' solution). For observations a Siemens Elmiskop was employed.

Results

A. Ommatidial Morphology Th e s t ru ctu r e of an o m m a t i d i u m in Aeschna's c o m p o u n d eye is of t y p i c a l apposition type, a n d is d i a g r a m m a t i c a l l y shown in Fig. 1 based on t h e present electron microscopical observations. Thus t h e o u t e r m o s t element, t h e corneal lens is p r o d u c e d by th e secretion of two Semper cells located j u s t b e n e a t h t h e cornea. T h e n e x t dioptric layer is m a d e up of crystalline cones, each developed b y four crystalline cone cells l o cat ed peripherally a r o u n d t h e cone, which shows four parts in cross section. Th e crystalline cone cells e x t e n d th ei r p r o x i m a l processes deep into the r e t i n u l a r l a y e r as far as t h e b a s e m e n t m e m b r a n e . Th e posi-

Structure and Spectral Sensitivities of Dragonfly Eye

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Fig. 1A-D. Diagrams of an ommatidinm from an Aeschna cyanea compound eye. A Longitudinal view. (Cr) corneal lens, (Cc) crystalline cone, (Cp) the proximM processes of crystalline cone which reach the basement membrane (Bin) along the rhabdom (Rh), (Rc) retinular cells, (Ra) Rc axons. B Cross section through the distal retinula. The eight retinular cells are numbered from one to eight and the three part rhabdom is evident; the rhabdomere of retinular cell 1 contributes one part, those of 3 and 4 another and those of 6 and 7 the third. The four filled circles represent the proximal processes (Cp) of the crystalline cone. C Cross section of the middle layer of a retinula which resembles (B) except for the absence of mierovilli on retinular cell 7. D Cross section of the proximal retinular layer showing the two part rhabdom composed of rhabdomeres from proximal retinular ceils 2 and 5

t i o n s of t h e s e p r o x i m a l processes a r e c o n s t a n t for all o m m a t i d i a as s h o w n in Fig. 1. T h e y a r e l o c a t e d b e t w e e n r e t i n u l a r cells 1 a n d 2, 3 a n d 4, 5 a n d 6, 7 a n d 1. S e v e r a l p i g m e n t cells s u r r o u n d t h e c r y s t a l l i n e c o n e a n d f u n c t i o n as a l i g h t shield.

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The retinular layer comprises a single layer of very slender retinular cells extending from the end of the crystalline cone to the basement membrane, t~etinular cell axons penetrate the basement membrane and course to the lamina. For each ommatidinm the retinular layer consists of eight retinular cells: five of these are dominant and have their nuclei in the distal part of the retinular layer. Two other retinular cells are dominant and have their nuclei in the very proximal part of the retinular layer whereas the last one is very small located peripherally in the ommatidinm and has no rhabdom mierovilli. For convenience, the eight retinular cells in an ommatidium are numbered in this paper according to the positions of the four crystalline cone processes (Fig. 1). Thus the retinular cell which has cone processes on each side is No. 1, and the other cells are numbered so that the rudimentary cell is No. 8. The longitudinal section through the rhabdom (Fig. 2) shows a typical insect rhabdom structure, and does not show the banding structure seen in crab eye. The retinular layer can be divided into three snblayers according to the numbers of rhabdomeres. In the most distal quarter of the retinula the rhabdom is composed mainly of five rhabdomeres belonging to retinular cells 1, 3, 4, 6 and 7 and their corresponding cell bodies. The other retinular cells, No. 2, 5 and 8 are very small here appearing in sections as flattened processes (Fig. 3). In this distal layer the rhabdom of each ommatidium shows three parts each with the direction of its mierovHli separated by about 120 ~ The rhabdomere of cell 1 contributes one of these three directions, those of 3 and r which are both of the same size comprise those microvilli with the second orientation, whereas those of retinular cells 6 and 7 contribute the third direction with 6 making up a greater part than 7. In the middle layer (Fig. 4), the rhabdom's architecture is almost the same as that of the distal layer described above, except for the fact that retinular cell 7 has no mierovilli, and is smaller and more slender like the distal process of proximal retinular cells 2 and 5. I n the proximal layer (about a quarter of the total retinula length), the rhabdom (Fig. 6) is quite different in structure than in the distal and middle layers. Proximally, most of the retinular volume of an omma-

Fig. 2. Longitudinal section through the rhabdom of a dragonfly ommatidium showing the regularly arranged mierovilli. (V) perirhabdomal vacuole, (B) multivesicular body, (M) mitochondrion. • 26000 Fig. 3. Cross section through the distal retinula showing the three part rhabdom, eight retinular cells numbered one to eight, and four proximal processes of crystalline cone cells (arrows). x 25000

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tidium is occupied by the proximal retinular cells 2 and 5 which are not prominent in the distal and middle layers. The corresponding part of the rhabdom is composed of only the rhabdomeres of these two proximal retinular cells. Their respective mierovilli make an angle to one another of about 120 ~ (or 240~ The direction of the mierovilli in proximal retinular cell 2 is the same as that of 6 and 7 in the distal and middle layer, and that of 5 is the same as that of 1. I n this layer the rhabdom lacks one of the three directions of mierovilli, namely that of retinular cells 3 and 4. Retinnlar cell 8 has no mierovilli and does not share in rhabdom formation anywhere along the retinula so far as observed in this experiment. Consequently this cell may not function in visual excitation. Nevertheless, it sends its proximal axon to the lamina through the basement membrane. Distally the proximal retinular cells (2, 5) have ribbonlike flattened processes extending out to the distal margin of the retinnlar layer. They run between the distal retinular cells and their specialized edges come in close contact with the microvilli in the distal and middle rhabdom layers. In these regions of intercellular contact there are areas resembling tight junctions where the intercellular space, normally about 200 A or more between retinular cells, narrows down to 100 A or less (Fig. 5). Summarizing, the distal retinular cells 1, 3, 4, 6 and 7 have their nuclei in the distal layer and form the rhabdom in the distal and middle layers. They become slender in the proximal layer and send their axons centrally to the lamina. On the other hand, the proximal retinular cells 2 and 5 have the main part of their cell bodies including nuclei in the proximal layer, and send their distal processes peripherally and their axons centrally (Fig. 7). Desmosomes between membranes of two retinnlar cells are seen near the rhabdom in cross sections. Mitochondria are mainly restricted to the cytoplasm near the rhabdom. I n the spider crab, Libinia, thelight adapted eye contained significantly more cytoplasmic organelles such as multivesieular bodies, pinoeytotie vesicles, etc. in retinular cell cytoplasm than the dark adapted eye

Fig. 4. Cross section through the middle retinular layer showing the three part rhabdom, eight retinular cells and four proximal processes of crystalline cone (arrows). • 12000 Fig. 5. Detail of a middle retinular layer cross section showing part of the rhabdom and the distal process of a proximal retinular cell (P). Its terminal edge (arrow) extends to the rhabdom and makes close contacts with mierovillus membranes. • 62000

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(Eguchi and Waterman, 1967). These results are confirmed in experiments on light and dark adapted Aesehna eyes.

B. Spectral Sensitivities I. Measured with Fixed Stimulus Direction I n about half of the electrophysio]ogical experiments, the angle of the stimulus light was fixed, and the eye preparation was mounted so that the longitudinal axis of the ommatidium and the light beam of the stimulating light coincided. From each retinular cell recorded, response-energy relations were measured for the different light intensities at wavelength 392 nm, and spectral sensitivity recordings were continued as long as the amplitudes of the response at 392 nm stayed constant. The resulting spectral response curves (in mV) were transformed to spectral sensitivity curves (in %) by using response-energy curves in the manner introduced by Autrum and yon Zwehl (1964). In all the 53 units were held sufficiently to measure both their response-energy curves and spectral sensitivities. The results can be grouped in five different types: (1) UV type, (2) blue type with a single peak at )~max445-458 nm in the visual range (400-615 nm), (3)green type with a single peak at 2m~x 475-519 nm in the visual range, (4) shifting type with a 2max shifted from type 2 to type 3 or vice versa during m a n y runs of recording, (5) double type having two peaks (type 2 and type 3) in a single spectral sensitivity curve. The more detailed characteristics of these types are the following. 1. U V Type. Single peak in the ultraviolet range Xmax at 356 nm and no secondary peak in the visual range (Fig. 8). NTumbcr observed : 3. 2. Blue Type. Main peak at 2m~x 445-458 nm and a secondary peak at 371 nm (Fig. 9). The theoretical resonance curve for a visual pigment 2max 458 (dotted smooth line in Fig. 9, Dartnall, 1953) does not fit this spectral sensitivity curve. Number observed: 18. 3. Green Type. Main peak at 475-519 nm, secondary peak at 372 nm (Fig. 10). The theoretical resonance curve at 2max 494 nm (dotted smooth line in Fig. 10) fits the spectral sensitivity curve well between 400-574 nm. Number observed: 12.

Fig. 6. Cross section through the proximal retinular layer showing 7 retinular cells numbered one to seven and the two part rhabdom composed of rhabdomeres from proximal retinular cells 2 and 5. • 24000 Fig. 7. Cross section just beneath the basement membrane showing eight retinular cell axons from an ommatidium. • 6600 14

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4. Shi/ting Type. 2max shifts r e p e a t e d l y f r o m t y p e 2 to t y p e 3 a n d vice v e r s a (Fig. l l ) . N u m b e r o b s e r v e d : 10. 5, Double Type. T w o m ~ i n p e a k s (like ~ype 2 a n d t y p e 3 superi m p o s e d ) (Fig. 12). N u m b e r o b s e r v e d : 10.

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II. Measured with Adjustable Stimulus Direction In the experiments described above, the microeleotrode was inserted into the middle layer of the retinula in such a way that there was no assurance that direction of the stimulus and the optic axis of the cell being recorded coincided closely. Conceivably the shifting and double 14"

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213

type units reported above were the result of this stimulus obliquity. Therefore in the following experiments, the angle of the stimulating light was freely adjustable by means of a bundle of flexible glass fibers acting as a light guide. B y this means the relation between the response amplitude and the angle of stimulus incidence was studied in the green type retinular cell with constant light intensity (Fig. 13). Incoming light was found to stimulate the retinular cells within a very narrow angle (8 ~) (Fig. 13). Initially the light direction was adjusted parallel to the long axis of the ommatidium to maximize the response to a constant light intensity. Then three series of the spectral sensitivities were measured (Fig. 14A): these are identical to the green type with 2max 475-495 nm already described. Then the angle of stimulus incidence was altered by 4 ~. This caused the responses of the retinular cells to decrease by about one third from the m a x i m u m responses with 0 ~ deviation (Fig. 13). Furthermore the non-axiM light incidence significantly altered the apparent spectral sensitivities (Fig. 14B). Of three runs made two of them are of the "double t y p e " and show the peaks at 458 nm (blue type) and at 519 nm (green type). The third run showed a "shift t y p e " response, and has its peak at ~max 458 nm with a little shoulder between 495-539 nm instead of the )~max of 475-495 nm observed with the parallel incidence. After these three runs, the stimulus angle was again set back to parallel the long axis of the retinnlar cell. Then three more runs of spectral sensitivities were measured. The results (Fig. 14 C) again showed the green type with 2max at 475 nm in the visual range which is identical to t h a t of Fig. 14A. The secondary peak at 371 nm was not affected by the angular deviation of the stimulus light and stayed constant throughout the whole experiment of nine runs. This experiment demonstrates that the "double t y p e " and the "shift t y p e " of element m a y not be independent types of cell but are derived from the green type b y an artifact arising from a deviant stimulus angle.

Discussion In general the present evidence for distinct proximal and distal groups of retinular cells in Aeschna confirms the pattern derived from light microscopy (Zimmerman, 1941). Furthermore the electron micrographs show t h a t the Aeschna rhabdom, like t h a t of other arthropods, is made up of regularly arranged groups of closely parallel mierovilli. At least in some cases the biological meaning of the single directional orientation of the mierovilli in one rhabdomere is related to the capacity of arthropod and cephalopod eyes to analyze polarized light (Eguehi and Waterman, 1966; Shaw, 1966; Tasaki and Karita, 1966; W a t e r m a n and Horch, 1966).

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E. Eguehi: Structure and Spectral Sensitivities of Dragonfly Eye

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Three dh~eetions of mierovilli orientation have been previously observed in other dragonfly eyes in addition to Aeschna. Thus Agriocnemis (Naka, 1961), Sympetrum (ttorridge, 1969) and damsel flies Ishnura and Cereion (Ninomiya, Tominaga and Knwabara, 1969) all show a three part rhabdom, each with mierovilli arranged at angles of about 120 ~ to one another. Therefore in dragonflies information on e-vector orientation in polarized light would appear to be analyzed in a three channel system instead of the two orthogonal channels observed in many crustaceans, cephalopods and certain insects (e. g. Apis, Goldsmith and Philport, 1957). There are, however, some reported differences in the number of retinular cells bearing rhabdomeres in different dragonfly species. Thus Aesehna has eight whereas in Sympetrum there are five (tIorridge, 1969) and in Agriocnemis only four (Tominaga, cited by Naka, 1961). On the basis of e-vector sensitivity the three channels mentioned above are partitioned among the seven rhabdomere bearing cells as follows : 1. retinular cells 1 and 5, 2. cells 3 and 4, and 3. cells 2, 6 and 7. Because the rhabdomeres of the proximal retinular cells (2 and 5) extend from the basement membrane through only about 20 % of the total rhabdom length, they presumably require for excitation more closely axial light rays than the distal retinular cells. According to the present electrophysiological data the widest acceptance angles were 7-8 ~ presumably for the distal cells. One m a y surmise that the deeper lying proximal cells have visual angles limited to about 1 ~ although no direct data are yet available on this point. Another interesting feature of the proximal cells is that the distal processes make close junctions with rhabdomeres of other retinular cells. The relationships suggest information transfer between the proximal and distal parts of the retinula but its existence and significance remain to be demonstrated. The function of the eighth retinular cell is obscure. Because it lacks microvilli, it may be considered nonfunctional in light reception. But since it does send an axon to the lamina, it may yet be significant in visual information processing. The present demonstration in the Aeschna eye of units having two absorption peaks in the visible range raises interesting problems. Fig. 14A-C. The effect of the angular deviation of stimulus direction on the spectral sensitivity curves recorded in 9 successive runs with a single green receptor. A Three spectral sensitivity runs with ~max at 475~195 nm to light just parallel to tile long axis of the ommatidium. B The next three spectral sensitivity runs with the light direction deviant by an angle of 4 ~ to the ommatidium axis; Areax 458 nm and 519 nm. C The next three spectral sensitivity runs with the light direction parallel to the optical axis of the ommatidium "~maxat 475 nm

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Although some insect's retinular cells yield single peak sensitivity curves (e. g. Apis and Calliphora) others have been found with double peaked responses, also (e.g. in Locusta, Bennett, Tunstall and Horridge, 1967). If this indicates that two visual pigments are present in a single receptor cell, as was concluded to be the case for Locusta, the functional significance of this situation is unclear. The presence of more than one visual pigment in a retina is usually related to scotopic-photopic specialization or to color discrimination. In the latter function at least, two or more photopigments having different ~r~x's must be in different cells according to Johannes Miiller's old doctrine of specific nerve energies. Thus in the fish retina each of three types of cones normally has a single peak in the visual range although the blue receptor may contain some red pigment (Marks, 1965; Tomita, Kaneko, Murakami, and Pautler, 1967). Several alternative explanations might account for the two peaks observed in a single Aeschna retinal element. Thus the electrode tip may be so placed that it simultaneously records from two contiguous cells (Autrum and yon Zwehl, 1964; Waterman and Ferns 1970). Wavelength sensitive nervous inhibition acting through some feedback mechanism is another possibility but there is no relevant evidence. Selective absorption by dioptric elements like the cornea and crystalline cone might have an appropriate influence. But microspeetrophotometric measurements of these components in Aeschna show no significant absorption bands between 300 and 540 nm (Ko]b, Autrum, and Eguchi, 1969). However, the experiments described above using the fiber bundle directional light stimulus demonstrated that the presence of two peaks was dependent on the stimulus direction being oblique to the ommatidial axis. When axis and direction coincided, the response was that of a single peaked green receptor. These directional effects might result from the absorption spectrum of a screening pigment which is leaky over part of the visible range (Burkhardt, 1962; Goldsmith, 1965; Waterman and Ferns 1970; Woodcock and Goldsmith, 1970 in press). However, the screening pigment in these previously known eases is leaky at the longer wavelengths so that oblique light incidence shifts sensitivity towards the red. In the present experiments obliquity of the stimulus induced a second peak at shorter wavelengths (458 rim) than the axial green Xmax (519 rim). Furthermore spectrophotometric measurements of pigment granule absorption from the eyes of several insects indicates that they are neutral filters without any significant selective absorption between 320 and 590 nm (Langer, 1967). Thus further research is required to explain the striking correlation of stimulation direction and the two peak responses in Aeschna retinular cells. The same is true for the shifting green-blue alternate peak units frequently recorded. At present they should probably be considered as green


217

units whose response peak can be shifted by some as yet unknown mechanism. We m a y conclude then that the dorsal region of the compound eye of

Aeschna has only two types of color receptors, an ultraviolet type and a green type. The green type occurs also in the ventral part of Aeschna's eye (Autrum and Kolb, 1968). But the blue violet type (2m~x 412~I32 nm) observed in the ventral region is not present in the dorsal half; on the other hand the UV receptor in the dorsal part has not been found in the ventral area. The different distributions of these three color receptors in the dorsal and ventral halves may be closely associated with their functions since the dorsal part of a compound eye of Aeschna looks mainly at the sky and the ventral part looks at the ground. In Libellula the dorsal part of the eye has only one type of color receptor with a 2max at 420 nm determined by the colorimetric methods (Mazokhin-Porshnyakov, 1957). On the other hand, the compound eye of Aeschna has three different types of color receptors. The two-type system (UV and green) found in the dorsal part of Aeschna's eye is quite similar to that found in the cockroach Periplaneta compound eye (Mote and Goldsmith, 1970). By comparing electron microscopic observations with the electrophysiological results some suggestion may be made on the possible arrangement of the UV and green receptors among the eight retinular cells in an ommatidium. Since retinular cells 1, 3, 4, and 6 occupy the greater part of the retinula in an ommatidinm in the distal and middle layers, the probability that the tip of an electrode was inserted into one of these distal retinular cells is much greater than for the proximal cells. Since only 3 of the total of 53 successful intracellular recordings were of the UV type, it would seem unlikely that these units lie in the distal retinular cell group. Therefore the distal retinular cells l, 3, 4, and 6 m a y be assumed to be green receptors. However, proximal retinular cells 2 and 5 and one distal retinular cell (No. 7) occupy much less retinular volume than the distal retinular cells. Consequently the penetration of these cells by the vertically inserted micropipette is proportionately less likely for these proximal retinular cells. These facts suggest that proximal retinular cells 2 and 5 may be the UV receptors. Further work is required to categorize retinular cell 7. The author wishes to express his cordial thanks to Prof. Dr. It. Autrum and Dr. G. Kolb for their friendly help and encouragement. The author is greatly indebted to Prof. Dr. D. Schneider, Max Planck Institut fiir Verhaltensphysiologie, Seewiesen, for generously sharing his electron microscopic facilities. The author is also grateful to Miss Mfiller and Miss Thies in the Max Planck Institut and Mrs. Barth, Miss Sorge and Miss H611dobter in Zoologisches Institut tier UniversitEt Miinchen for their technical assistance. The author wishes to thank Dr. T. H. Waterman, Biology Department, Yale University, New Haven, Conn., U.S.A., for his review and criticism of this manuscript.

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E. Eguchi: Structure and Spectral Sensitivities of Dragonfly Eye

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