Quantitative Studies on Pigment Migration and Light Sensitivity in the Compound Eye at Different Light Intensities

C.G. BERNHARD and D. OTTOSON From the Department of Physiology,Karolinska Institutet, Stockholm, Sweden

ABSTRACT Comparative electrophysiological and histological studies were made on the functional significance of the secondary iris pigment migration for the sensitivity of the eye in the noctuid moth Cerapteryx grarninis. The pigment position at different adapting light intensities was studied as well as the influence of different positions on the sensitivity of the eye. Adapting light intensities above a certain value hold the pigment in light position. At a 3 log units lower intensity the pigment is brought into dark position and at light intensities between these limiting values the pigment attains intermediate positions. The results indicate that at light intensities between the limiting values the pigment shifts closely follow the changes in intensity of the environmental light. With the pigment in dark position the eye is about 1000 times more sensitive than when the pigment is in light position, there being a close relationship between the sensitivity of the eye and the position of the pigment at intermediate positions.

In retinae containing pigment which migrates under the influence of light (Garten, 1907) the temporal course of dark adaptation is likely to be more or less influenced by the positional changes of the migratory pigment. In arthro- pods the eyes show a great variety of pigment arrangements (Parker, 1932), such as intraretinular pigment, basal pigment in the region where the nerve fibers penetrate the basement membrane, primary iris pigment in distal pigment cells situated around the crystalline cones, and finally, secondary iris pigment within large proximal pigment cells which like wedge-shaped pillars surround the crystalline cones and the translucent filaments connecting the crystalline cones and the rhabdome. The organization of the intraretinular pigment in the Limulus eye has been described in recent investigations by Miller (1958) who showed radial displacements of the pigment granules due to illumination. A complex form of compound eye like that of Astacusfluviatilis, for example, exhibits marked longitudinal displacements of both the basal and the retinular pigment as well as of the iris pigment (Bernhards, 1916). The

465

The Journal of General Physiology 466 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 47 " I964 basal pigment in day-flying insects, e.g. butterflies, shows very slight movements in response to illumination (see Demoll, 1909), and the same seems to be the case for the distal pigment of the butterflies (Demoll, 1909; Bernhard and Ottoson, 1960 a). As judged by recent investigations on Sympetron (Bern- hard, H6glund, and Ottoson, 1963), these movements do not seem to have any significant influence on the sensitivity curve during dark adaptation. In the eyes of the noctuid moths, which, besides having the primary iris pigment and basal pigment are equipped with secondary iris pigment, a very marked longitudinal migration of the secondary pigment occurs under the influence of light (Wigglesworth, 1953). In recent investigations it has been shown that the compound eye of night moths offers an excellent model for a study of the influence of pigment migration on the sensitivity of the eye (Bernhard and Ottoson, 1959, 1960 a and b, 1961, 1962; Bernhard, H6glund, and Ottoson, 1963). In these investigations the relation between the position of the secondary iris pigment and the changes in threshold of the retinal potential during the course of dark adaptation was studied in night moths. A biphasic dark adaptation curve was found (Bernhard and Ottoson, 1959, 1960 a) and it was shown that there is a correlation between the second phase of the dark adaptation curve (see Fig. 1 A) and the positional changes of the secondary iris pigment from light position (see Fig. 1 C) to dark position (see Fig. 1 D) during dark adaptation. This type of dark adaptation curve could also be demonstrated in other species equipped with secondary iris pigment of the same type as that of the noctuid Lepidoptera but was never found in the compound eyes of day-flying insects lacking secondary iris pigment. When, as a consequence of strong illumination of long duration or due to impairment of the preparation by the dissection procedure, the migration was blocked so that the pigment remained in light position, the second phase of the dark adaptation curve was not obtained (Bernhard and Ottoson, 1959, 1960 a; Bernhard, H6glund, and Ottoson, 1963). It was also found that a prerequisite for the appearance of the second phase is that the pigment be brought into light position before letting the eye dark-adapt; i.e., that this phase is lacking when dark adaptation follows illumination of too short duration to bring the pigment into light position (H6glund, 1963). It has also been found that there is not always a clear temporal separation of the two phases of the dark adaptation curve. Thus, due to the fact that the latency of the pigment migration in many species is short, the two phases may be fused (Bernhard, H6glund, and Ottoson, 1963). Among the different species of the night moths so far tested, Cerapteryx graminis offered the most favorable preparation for the study of the relation between the positional changes of the secondary pigment and the sensitivity to light during dark adaptation because of the clear temporal separation of the two phases of the adaptation curve (see below and Fig. 1 A). Therefore this species was also used in the investigations to be presented. C. G. BERNHARD AND D. OTTOSON Pigment Position and Sensitivity of Moth's Eye 467

The aim of the present investigation was to obtain further information concerning the influence of migration on the sensitivity of the eye by studying the position of the pigment and the light sensitivity at different adapting light intensities.

MATERIAL AND METHODS The experiments were carried out on excised heads of fresh specimens of the night moth Cerapteryx graminis. The procedure used for leading off the electrical retinal response and the electrophysiological and illumination equipment were the same as used

FIGtJr~E 1. A, average dark adaptation curve of Cerapteryx graminis (Bernhard, H6glund, and Ottoson, 1963). B, threshold changes during exposure to dim light of relative intensity - 3.4 followed by darkness. In- sets show sections of the eye in light-adapted (C) and dark- adapted (D) state. in previous investigations (Bernhard and Ottoson, 1960 a; Bernhard, H6glund and Ottoson, 1963). The histological technique used has also been described earlier (see Bernhard and Ottoson, 1961). Light intensities are expressed in log units.

RESULTS Fig. 1 A shows the average dark adaptation curve of Cerapteryx graminis after 3 minutes' exposure to bright light with a relative intensity of - 1 (Bernhard, H6glund, and Ottoson, 1963). There is at first a rapid fall in the threshold which according to our earlier investigations shows the post-illuminatory threshold changes with the pigment in light position (see inset C in Fig. 1). 468 THE JOURNAL OF CENERAL PHYSIOLOGY • VOLUME 47 " I964

When the light is turned off the pigment remains in light position for, on the average, 7 minutes. After this conspicuously long latency the pigment starts to retract and slowly moves into dark position (see inset D in Fig. 1) during the following 20 to 30 minutes (Bernhard and Ottoson, 1959, 1960 b; Bern- hard, HSglund, and Ottoson, 1963). At the same time the second phase of

-1

-2

-3-

-Sno L

o~ .J

-5

-6

-7

Ad. Light Log I-4 Darkness IIm

-8 I I t I [ 10 20 30 40 50 Time (mini FmURE 2. Threshold changes during exposure to dim light of relative intensity -4 followed by darkness. C. O. BERNHARD AND D. OTTOSON Pigment Position and Sensitivity of Moth's Eye 469 the dark adaptation curve develops and the fall in threshold shows that the pigment retraction is followed by an increase in sensitivity of about 2 to 3 log units. As mentioned in the introduction the duration of the plateau is depend- ent on the experimental conditions used as regards intensity and duration of the preadapting light. Curve B in Fig. 1 shows the sensitivity changes during exposure of the eye to light with a relative intensity of -3,4 after preadaptation to bright light.

-2

-3

"O "5 -~ -4-

o ~o

-5-

-6

Ad. Light Log I-4.6

"V-----.----O-

, t -7 ,o 2'0 3; 40 s0 Time (mln.) FIouRE 3. Threshold changes during exposure to dim light of relative intensity -4.6 followed by darkness.

As seen, no second phase appears, the threshold remaining at the plateau level during 18 minutes, i.e., as long as the adapting light was on. That the lack of the second phase is not due to impairment of the eye is shown by the fact that there is a sudden fall in the threshold as soon as the adapting light is turned off and the eye is kept in darkness (marked by the black horizontal column at the bottom of the diagram). On the basis of the results referred to 47 ° THE JOURNAL OF OENERAL PHYSIOLOGY • VOLUME 47 • ~964 above the conclusion would be that the relative intensity of the adapting light (-3.4) was not low enough to allow any positional change of the pigment. It should also be noted that, whereas the second phase of the dark adaptation curve has an average latency of 7 minutes when the eye had been kept in bright light (relative intensity -1), the fall in threshold appears without

o ..-I -5-

-6-1 Ad. Light Log I - 5.2 Darkness

-7 I i 10 20 3'0 Time (rain,) Fm~mE 4. Threshold changes during exposure to dim light of relative intensity -5.2 followed by darkness. appreciable delay when darkness follows after an adapting light of lower intensity, as shown by curve B. The conclusion would be that the pigment retraction tends to start after a considerable delay after exposure of the eye to bright light, whereas the pigment migration begins without appreciable delay when the eye has been exposed to a lower light intensity, in this case a relative intensity of - 3.4. As seen in Fig. 2 an exposure of the eye to light with a slightly lower intensity (relative intensity -4) after preadaptation to bright light was not followed C. Car. BERNHARDAND D. OTTOSON Pigment Position and Sensitivity of Moth's Eye 471 by any second phase either. When the dim light was turned off there was an instantaneous further fall in threshold. The diagram in Fig. 3 shows that by using an adapting light of slightly lower intensity (relative intensity -4.6) the second phase of the dark adaptation curve was obtained. The second fall, however, only covers less than one log unit which would indicate that at this intensity of the adapting light there is only a partial pigment retraction. Dark-

-2-

-3-

"0 o

t-'

o ~o .-I

-5

-6 Ad, Light Log I - 6.4 ~Darkness

-7 I I ! ! 0 20 30 40 Time (rain.) FIGURE 5. Threshold changes during exposure to dim light of relative intensity -6.4 followed by darkness. ness is then followed by the usual fall in threshold indicating completion of the pigment retraction. The lower the intensity of the adapting light, the more pronounced is the second phase as seen in the next two figures (Fig. 4, relative intensity -5.2; and Fig. 5, relative intensity -6.4). Finally, when the eye is exposed to light with the relative intensity of -7 after preadaptation to bright light, an opti- mal second phase develops (Fig. 6). Thus, the turning off of this dim light is 472 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 47 • 1964 not followed by any further fall in the curve, which would indicate that full pigment retraction occurs at this low light intensity as in total darkness. The graph in Fig. 7 shows the relation between the final threshold values obtained during the second phase of the adaptation curves and the various adapting light intensities used. As mentioned the threshold curves obtained

-1

-2

-3

' 4

..J

-5

-6

-7

Ad. Light Log I-7 Darkness

-8 1'0 20' 3 0 40 ' Time (rain.) FIGUI~ 6. Threshold changes during exposure to dim light of relative intensity - followed by darkness. C. G. BERNHARDAND D. OTTOSON Pigment Position and Sensitivity of Moth's Eye 473 at adapting light intensities above -4 did not show any second phase. At adapting light intensities between -4 and -7 the second phase of the adaptation curve increased successively and intensities below -7 were not followed by any further increase of the second phase.

-4

v o~ _J -6

-7

I I I I I I 3 -4 -5 - 6 -7 -8 Adapting Light Log I 17IGURE 7. Relation between adapting light intensity and final threshold during second phase of adaptation.

I.I 100

so E

| -8 Adapting Light Log I FIOUR~ 8. Pigment retraction at different adapting light intensifies. 474 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 47 • 1964

From the results presented above the conclusions would be (a) that in order to start pigment retraction from the light position, the eye has to be exposed to light with intensities below a certain value (in our experiments relative intensity -4) and (b) that full pigment retraction is obtained at a certain intensity (relative intensity around -7). Exposures of the eye to intensities between these values would thus be expected to result in intermediate pigment positions. In order to test the validity of these conclusions, experiments were made in which the position of the pigment was studied histologically after the eyes had been exposed for 40 to 50 minutes to adapting light of different intensities.

b -4

-o -5 -6

,&, o~

-7

| I i I I I I I I I 50 ]00~ Pigment Retraction Fm~E 9. Threshold intensities at different stages of pigment retraction from light- adapted position (zero on abscissa) to maximal dark position.

The results of such a study are presented in Fig. 8 in which the position of the proximal border of the pigment in relation to light position is plotted in microns against the various intensities of the adapting light used. As seen, the pigment stayed in light position at intensities above -4, intermediate positions were found between -4 and -6.6, and at intensities below -7 the dark position was obtained. Fig. 9, finally, shows the relative sensitivity of the eyes at various pigment positions. The logarithm of threshold intensity is plotted against position in microns of the proximal border of the pigment at positions between the light and dark position, the light position being taken as zero. A shift of the pigment from extreme light position to dark position covering on an average 90 to 100/z increases the sensitivity about 1000 times. If the sensitivity is logarithmically related to the position of the proximal border of the pigment, C. G. BERNHARD AND D. OTTOSON Pigment Position and Sensitivity of Moth's Eye 475 as suggested by the curve, it would mean that a pigment shift of 30 # towards dark position increases the sensitivity about 10 times.

DISCUSSION According to Tuurala (1954) the compound eye of Cerapteryx graminis is to be regarded as a typical noctuid superposition eye with iris and basal pigment cells and no intraretinular pigment. With the histological methods used no significant positional changes of the basal pigment could be seen under the influence of light, whereas there were pronounced longitudinal migratory movements of the secondary iris pigment (see Fig. 1 C and D). In light position the pigment surrounds the translucent filaments connecting the crystalline cones and the retinular cells and in dark position it is retracted proximally into the spaces between the crystalline cones together with the primary iris pigment. The proximal border of the secondary iris pigment moves on the average 90 ~ from light position to dark position. It is known that in the night moth eye, the position of the secondary iris pigment varies according to the intensity of the light to which the insect is subjected, as shown by Collins' (1934) investigations on the codling moth (cJ. Day, 1941, on Ephestia kuehniella). Our histological studies on Cerapteryx graminis show that at light intensities above a certain value (relative intensity referred to as -4) the pigment remains in light position and that below a certain value, representing about 3 log units lower intensity, it stays in dark position. The diagram in Fig. 8, showing the relation between the degree of pigment retraction and adapting light at intermediate intensities, indicates the precision with which the iris pigment attains varying positions depending on the intensity of the environmental dim light. It has earlier been shown on the same species that there is a close correlation between the post-illuminatory threshold and the pigment position during the second phase of the dark adaptation curve (Bernhard and Ottoson, 1959, 1960 b, 1962; see Fig. 1). The functional significance of the migration of the secondary iris pigment for the stimulating power of the illumination is also illustrated by the diagram in Fig. 9 showing the relation between the degree of pigment retraction and the threshold for response at adapting light intensities between the relative intensities -4 and -7, at which the pigment is brought into varying intermediate positions. It is likely that the vital activities of this species at the light intensities above, between, and below these two limiting values are different. According to investigations on the codling moth the state of adaptation is a prominent factor in the behavior of the insects (Collins, 1934). It has been assumed that the migration of the secondary iris pigment merely effects a variation of the light which is transmitted to the photosensitive parts of the receptor ceils, In this context it should be noted that the pigment surrounds the transparent filaments distal to the region of the photosensitive 476 THE JOURNAL OF GENERAL PFIYBIOLOGY • VOLUME 47 • 1964 parts of the retinular cells. If the translucent filaments are assumed to possess wave guide properties, positional changes of the pigment surrounding the filaments may change these properties. Actually, Kuiper (1962) has suggested that the pigment system of the type described acts as a longitudinal pupil. As also pointed out by Kuiper (1961) the data obtained in studies on the relation between pigment migration and light sensitivity (Bernhard and Ottoson, 1960 b) are in agreement with such a theory. As to the question concerning a humoral and/or nervous or direct regula- tion of the pigment movements, different opinions have been expressed. In certain crustaceans the retinal pigment movements are hormonaUy governed by an eye stalk gland (Kleinholz, 1936, 1938; Welsh, 1939, 1941). To the authors' knowledge no such regulating mechanism has been found in insects. Negative results on night moths have been reported by Day (1941) on the basis of injection experiments with sinus gland extracts on F_,phestiakuehniella. Day (1941) concluded that the positional changes are controlled by a nervous mechanism. According to him the migration into dark position cannot be induced in Ephestia after transection of the optic tract. On the other hand Tuurala (1954) reports that iris pigment migration can be induced in the eyes of Tolera and F/phestia after resection of the ganglion and concludes that the positional changes of the iris pigment due to illumination are mediated independently of neuronal and humoral influences. This finding has been confirmed by H6glund (1963) who also demonstrated that the pigment migrates after the ganglionic activity is blocked by nicotine or cocaine. The fact that after complete damage of the regulatory mechanisms of the pigment migration by dissection (Demoll, 1911; Day, 1941), narcotics (Day, 1941), or "overillumination" (Bernhard and Ottoson, 1960 b, 1962), the pigment stays irreversibly in light position indicates that the movement into dark position represents an active phase. In this context it should be noted that in Cerapteryx graminis the pigment migration has a surprisingly long latency after illumination with bright light and this observation does not speak in favor of a simple nervous control. It is also interesting to note that after preillumination with light of lower intensities the sensitivity changes due to pigment retraction start without appreciable delay (Fig. 1-5). Ob- viously the positional changes of the pigment readily follow slight variations in the brightness of the environmental light at intensities at which this insect is expected to exhibit its highest vital activity.

This work has been supported by grants from the Office of Scientific Research of the Air Research and Development Command, United States Air Force through its European Office (contract AF 61-052-21) and from Stiftelsen Therese och Johan Anderssons Minne, Stockholm. The investigations were carried out at the Mature field laboratory, Bj6rk6, Arholma, Sweden. Received/or publication, July 16, 1963. C. G. BERNHARD AND D. OTTOSON PigmentPosition and Sensitivity o/Moth's Eye 477

REFERENCES I. BERNHARD, C. G., H~GLUND, G., and OTTOSON, D., On the relation between pigment position and light sensitivity of the compound eye in different nocturnal insects, J. Insect Physiol., 1963, 9, 573. 2. BERNHA~, C. G., and OTTOSON, D., On the mechanisms of dark adaptation in day- and night-insects, Acta Physiol. Scand., 1959, 47, 383. 3. BERNHARD, C. G., and OTTOSON, D., Comparative studies on dark adaptation in the compound eyes of nocturnal and diurnal Lepidoptera, J. Gen. Physiol., 1960 a, 44, 195. 4. BERNHARD,C. G., and OTTOSON, D., Studies on the relation between the pigment migration and the sensitivity changes during dark adaptation in diurnal and nocturnal Lepidoptera, J. Gen. Physiol., 1960 b, 44, 205. 5. BERNHARD, C. G., and OTTOSON, D., Further studies on pigment migration and sensitivity changes in the compound eye of nocturnal insects, Acta Physiol. Scan&, 1961, 52, 99. 6. BERNHARD, C. G., and OTTOSON, D., Pigment position and light sensitivity in the compound eye of noctuid moths, Acta Physiol. Scand., 1962, 54, 95. 7. BERNHARDS, H., Der Bau des Komplexauges von Astacus fluviatilis, Z. wissensch. Zool., 1916, 116,649. 8. COLLINS, D., Iris pigment migration and its relation to behaviour in the codling moth, J. Exp. Zool., 1934, 69, 165. 9. DAY, M. F., Pigment migration in the eyes of the moth Ephestia kuehniella Zeller, Biol. Bull., 1941, 80, 275. 10. DF.MOLL, R., /]ber eine lichtersetzliche Substanz im Facettenauge, sowie fiber eine Pigmentwanderung im Appositionsauge, Arch. ges Physiol., 1909, 129, 461. 11. DEMOLL, R., Die Physiologie des Facettenauges, Ergebn. Zool., 1911, 2, 432. 12. GARTEN, S., Die Veriinderungen der Netzhaut dutch Licht, Graefe-Saemisch Handbuch der gesamten Augenheilkunde, Leipzig, W. Engelmann, 2nd edi- tion, 1907, 3, chapter 12, appendix. 13. H6GLUND, G., Glow, sensitivity changes and pigment migration in the compound eye of nocturnal Lepidoptera, Life Sciences, 1963, No 4, 275. 14. KLEINHOLZ,L. H., Crustacean eyestalk hormone and retinal pigment migration, Biol. Bull., 1936, 70, 159. 15. KL~INHOLZ, L. H., Studies in the pigmentary system of Crnstacea. IV. The uni- tary versus the multiple hormone hypothesis of control, Biol. Bull., 1938, 75, 510. 16. KuIPeR, J. W., The optics of the compound eye, Symp. Soc. Exp. Biol., 1962, 16, 58. 17. KmPER, J. W., personal communication, 196 I. 18. MILLER, W., Fine structure of some invertebrate photoreceptors, Ann. New York Acad. Sc., 1958, 74,204. 19. PARKER, G. H., The movements of the retinal pigment, Ergebn. Biol., 1932, 9, 239. 20. TUURALA,O., Histologische und physiologische Untersuchungen fiber die photo- 478 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 47 " I964

mechanischen Erscheinungen in den Augen der Lepidoptera, Ann. Acad. Sc. fenn. Series A, Biol., 1954, 24, 1. 21. WELSh, J. W., The action of eyestalk extracts on retinal pigment migration in the crayfish Cambarus bartoni, Biol. Bull., 1939, 77, 119. 22. WELSH,J. W., The sinus glands and 24 hour cycles of retinal pigment migration in the crayfish, J. Exp. Zool., 1941, 86, 35. 23. WmOL~SWORTH, V. B., The Principles of Insect Physiology, London, Methuen, 1953.