The Journal of Experimental Biology 202, 2539Ð2542 (1999) 2539 Printed in Great Britain © The Company of Biologists Limited 1999 JEB2206

CIRCADIAN OSCILLATION OF SENSITIVITY OF EYES: DIURNAL AND NOCTURNAL

SHIGEKI YAMASHITA* AND TAKIKO NAKAMURA Biological Laboratory, Kyushu Institute of Design, Shiobaru, Fukuoka 815-8540, Japan *e-mail: [email protected]

Accepted 3 June; published on WWW 25 August 1999

Summary The circadian oscillation of sensitivity of the anterior median eye of a nocturnal spider Araneus ventricosus and that of a diurnal spider Menemerus confusus were examined by recording electroretinograms. The anterior median eye of Araneus ventricosus showed a marked Key words: circadian sensitivity rhythm, anterior median eye, circadian oscillation of sensitivity, but that of Menemerus nocturnal spider, diurnal spider, Araneus ventricosus, Menemerus confusus showed no circadian oscillation. confusus, vision.

Introduction The anterior median eyes of the orb-weaving spiders intensity (logI=0). The duration of illumination was controlled bruennichii and Argiope amoena have three types of by an electromagnetic shutter, and the intensity was adjusted visual cells, with maximum sensitivities at approximately by calibrated neutral-density filters and wedges. A 360 nm (ultraviolet cell), 480Ð500 nm (blue cell) and 540 nm monochromatic light beam was produced using interference (green cell). The blue cells are the most sensitive and have a filters. The energy of the selected monochromatic light was circadian oscillation of sensitivity (Yamashita and Tateda, measured at the cornea of the eye using a radiometer. 1978). Argiope sp. appears to be active both during the day and Monochromatic light (520 nm) of 1014 quanta cm−2 s−1 and at night (a noct-diurnal spider); during the day, it stays in the white light of logI=−2 generated ERGs with a similar hub of its web and attacks its prey. In the present study, we amplitude. examined the circadian oscillation of sensitivity of the anterior median eye of the garden spider Araneus ventricosus and the Menemerus confusus. Araneus ventricosus Results constructs its web each day after sunset and destroys the web The anterior median eye of Araneus ventricosus showed a before sunrise, i.e. Araneus ventricosus is active only at night. marked circadian oscillation of sensitivity. An example is Menemerus confusus is a typical diurnal hunting spider, and a shown in Fig. 1. After the cessation of background number of its behavioural activities are initiated by visual illumination at 17:00 h, the ERG amplitude increased gradually stimuli (for reviews, see Forster, 1985; Land, 1985; Yamashita, for over 3 h. It then showed a circadian oscillation with a period 1985). of approximately 24Ð25 h under a constant dark background for 9 days. ERGs of constant low and high amplitude were recorded for approximately 9Ð11 h and 8Ð10 h, respectively. Materials and methods We call the former period ‘subjective day’ or briefly ‘day’, and The used in this study were female garden spiders the latter period ‘subjective night’ or briefly ‘night’. Araneus ventricosus and male and female jumping spiders Fig. 2 shows ERGs in response to white light stimuli of 0.1 s Menemerus confusus. They were collected in open fields. duration at various intensities recorded during the subjective Preparation and recording methods were similar to those day and night. The waveforms of night-time ERGs at logI=−7, described previously (Yamashita and Tateda, 1978). A −5 and −3 are similar to those of daytime ERGs at logI =−5, tungsten electrode was inserted into the eye to record −3 and −1, respectively. The intensity/response relationships electroretinograms (ERGs) from intact animals. For white light for daytime and night-time ERGs are shown in Fig. 3. The stimulation, light emitted by a Xenon arc lamp or a tungsten threshold intensity for the night-time ERG was approximately filament lamp was delivered to the eye via a quartz light guide, 2 log units lower than that for the daytime ERG. 0.2 mm in diameter, positioned in front of the corneal lens. The The spectral sensitivity of the anterior median eye was maximum intensity of the white light was referred to as unit examined by recording ERGs (Fig. 4). Under a constant dark 2540 S. YAMASHITA AND T. NAKAMURA

1 29 Aug Day Night 17:00 -5 -7 0 1 30 Aug -3 -5

0 1 31 Aug -3 -1 0 0.5 mV 1 1 Sep 0.2 s 0 Fig. 2. Electroretinograms (ERGs) in response to white light stimuli 1 2 Sep of 100 ms duration at various intensities obtained from the dark- adapted anterior median eye of Araneus ventricosus during the 0 subjective day and night. The intensity is indicated for each ERG in 1 3 Sep log units. Horizontal bars indicate 100 ms light stimuli. ERG amplitude (mV) 0 1 4 Sep 1.0

0 Night 1 5 Sep Day 0 1 6 Sep 0.5

0 15:00 18:00 21:00 00:00 03:00 06:00 09:00 12:00 15:00

Time of day (h) Relative ERG response

Fig. 1. Circadian changes in electroretinogram (ERG) amplitude obtained from the anterior median eye of Araneus ventricosus in a 0 constant dark background. The spider was collected in an open field -7 -6 -5 -4 -3 -2 -1 and maintained on a photoperiod of 12 h:12 h light:dark (light from LogI (relative intensity) 05:00 h to 17:00 h) for 2 days. After the cessation of the second 12 h light period at 17:00 h on 29 August, ERGs were recorded for 9 days. Fig. 3. Intensity/response relationships for the dark-adapted anterior A dim flash lasting 5 ms emitted by a light-emitting diode (560 nm median eye of Araneus ventricosus determined during the subjective emitting wavelength) placed in front of the preparation was day (open circles) and night (filled circles). The relative amplitude of automatically presented every 10 s. ERG amplitudes are plotted the electroretinogram (ERG) in response to white light is plotted every 10 min. The ERG amplitude between 20:10 h on 29 August against the relative stimulus intensity. The amplitude of the night- and 06:40 h on 30 August was greater than the limit of the scale of time ERG at LogI=−2 is referred to as 1.0. the recorder. background, daytime ERGs in response to a flash of No circadian oscillation of sensitivity was observed in the 1014 quanta cm−2 s−1 and night-time ERGs in response to a flash anterior median eye of Menemerus confusus. An example is of 1012 quanta cm−2 s−1 showed similar amplitudes at each shown in Fig. 5. After the cessation of background wavelength. Each curve had a broad peak at approximately illumination at 16:00 h, the ERG amplitude increased rapidly 520 nm in the visible region and a smaller peak or a shoulder within 1 min to a plateau level (Fig. 5A). The ERG amplitude in the ultraviolet region (approximately 360 nm). There was no then remained almost constant for approximately 1 month (Fig. significant difference between the daytime and night-time 5B). Similar results were obtained from the anterior lateral spectral response curves, as reported for Argiope spp. eye and the posterior lateral eye. The intensity/response (Yamashita and Tateda, 1978). Chromatic light adaptation did relationships obtained from the dark-adapted anterior median not change the spectral response characteristics. Fig. 4 shows eye at 10:00 h and at 20:00 h are shown in Fig. 6. The two the amplitude of the night-time ERG in response to a flash of curves are almost identical. The ERG threshold for Menemerus 1014 quanta cm−2 s−1 during adaptation to 540 nm light. These confusus was approximately 100 times higher than that for observations suggest that the anterior median eye of Araneus Araneus ventricosus during the subjective night (cf. Fig. 3). ventricosus has a single spectral type of photoreceptor that has We conclude that photoreceptor cells in the eyes of Menemerus a circadian oscillation of sensitivity. confusus do not show a circadian sensitivity rhythm. Circadian sensitivity oscillation of spider eyes 2541

2 3N A Dark-adaptation 2N 19 Oct

3D 2D ERG

3N LED 1 Back Light Dark Green- 16:00 h 16:10 h adaptation B

ERG amplitude (mV) 20 Oct 0 6 12 18

0 400 500 600 1 Nov Wavelength (nm) 0 6 12 18 Fig. 4. Spectral response curves of the dark-adapted anterior median eye of Araneus ventricosus for the subjective night (filled circles) 20 Nov and day (open circles), and that for the subjective night during 0 6 12 18 adaptation to 540 nm light (Green-adaptation, half-filled circles). The stimulus intensities for the dark-adapted eye during the subjective LED night and day are 1012 quanta cm−2 s−1 and 1014 quanta cm−2 s−1, respectively, and that for the light-adapted eye during the subjective night is 1014 quanta cm−2 s−1. The electroretinogram (ERG) 0.1 mV amplitude in response to a monochromatic stimulus is plotted against 3 min the stimulus wavelength. The ERG amplitude obtained from the Fig. 5. Electroretinograms (ERGs) of the anterior median eye of the light-adapted eye is magnified ten times, e.g. the actual value of the jumping spider Menemerus confusus. The spider was collected in an ERG in response to a 520 nm flash under the background light is open field and maintained on a photoperiod of 12 h:12 h light:dark approximately 0.12 mV. 2D, second subjective day; 2N, second (light from 04:00 h to 16:00 h) for 2 days. After the cessation of the subjective night; 3D, third subjective day; 3N, third subjective night. second light period at 16:00 h on 19 October, ERGs were recorded for approximately 1 month. A dim flash of duration 5 ms emitted by Discussion a light-emitting diode (LED; 560 nm emitting wavelength) was given every 10 s. (A) Changes in ERG amplitude after the cessation of 12 h Yamashita and Tateda (1978) reported that the more- light period at 16:00 h. (B) ERGs recorded on 20 October, 1 sensitive blue cells in the anterior median eye of noct-diurnal November and 20 November. The numbers indicate the time of day. spiders, Argiope spp., show a circadian oscillation of The occasional transient decreases in ERG amplitude may be caused sensitivity, but that the less-sensitive green and ultraviolet cells by eye movements (see Land, 1969). do not. As shown in the present study, the anterior median eye of a nocturnal spider, Araneus ventricosus, showed marked Circadian sensitivity changes in the eye that are controlled circadian oscillation of sensitivity, but the anterior median eye by efferent optic nerve fibres have been reported for Limulus of a diurnal spider, Menemerus confusus, did not. The posterior polyphemus (for a review, see Barlow et al., 1989), scorpions median eye and the posterior lateral eye of Argiope spp. demonstrate the morphological characteristics of noct-diurnal 1.0 spiders: i.e. they have two types of retina in the same eye (Uehara et al., 1978). In one type, the rhabdomic layer of the retina is backed by a tapetal reflecting layer. In the other type, the rhabdomic layer is backed by a pigmented layer. These Night observations suggest that Argiope is an intermediate type of Day spider between typical nocturnal spiders such as Araneus 0.5 ventricosus and typical diurnal spiders such as Menemerus confusus.

Fig. 6. Intensity/response relationships for the dark-adapted anterior Relative ERG response median eye of Menemerus confusus determined at 10:00 h (open circles) and at 20:00 h (filled circles). The relative amplitude of the electroretinogram (ERG) in response to white light is plotted against 0 the relative stimulus intensity. The amplitude of the night-time ERG -6 -5 -4 -3 -2 -1 0 at LogI=0 is referred to as 1.0. LogI (relative intensity) 2542 S. YAMASHITA AND T. NAKAMURA

Androctonus australis (for a review, see Fleissner and Fleissner, G. and Fleissner, G. (1988). Efferent control of Fleissner, 1988) and Argiope bruennichii and Argiope amoena visual sensitivity in eyes: with emphasis on (Yamashita and Tateda, 1981). Nakamura and Yamashita circadian rhythms. In Information Processing in Animals, vol. 5 (1997) recorded efferent impulses from the optic nerve of (ed. M. Lindauer), pp. 1Ð67. Stuttgart, New York: Gustav Fischer Araneus ventricosus. In Araneus ventricosus, efferent optic Verlag. nerve fibres may also control the sensitivity of the eye. The eye Forster, L. (1985). Target discrimination in jumping spiders (Araneae: Salticidae). In Neurobiology of (ed. F. G. of Menemerus confusus showed no circadian oscillation of Barth), pp. 249Ð274. Berlin, Heidelberg, New York: Springer sensitivity. We observed no efferent activity in the optic nerve Verlag. of Menemerus confusus, suggesting that Menemerus confusus Land, M. F. (1969). Movements of the retinae of jumping spiders may lack efferent optic nerve fibres. (Salticidae: Dendryphantinae) in response to visual stimuli. J. Exp. The eyes of spiders have various spectral types of receptor Biol. 51, 471Ð493. cells (for a review, see Yamashita, 1985). Walla et al. (1996) Land, M. F. (1985). The morphology and optics of spider eyes. In made intracellular recordings from photoreceptor cells of the Neurobiology of Arachnids (ed. F. G. Barth), pp. 53Ð78. Berlin, eyes of the ctenid spider Cupiennius salei, a nocturnal hunting Heidelberg, New York: Springer Verlag. spider, and found three spectral groups of cells with sensitivity Nakamura, T. and Yamashita, S. (1997). Phototactic behavior of maxima in the blue (480 nm), green (520 nm) and ultraviolet nocturnal and diurnal spiders: negative and positive phototaxes. (340 nm) regions. In the present study, we have demonstrated Zool. Sci. 14, 199Ð203. Uehara, A., Toh, Y. and Tateda, H. (1978). Fine structure of the that the anterior median eye of Araneus ventricosus has a eyes of orb-weavers, Argiope amoena L. Koch (Aranea: single spectral type of photoreceptor. It seems that the anterior Argiopidae). II. The anterolateral, posterolateral and posteromedial median eye of Araneus ventricosus is not capable of colour eyes. Cell Tissue Res. 186, 435Ð452. discrimination. Walla, P., Barth, F. G. and Eguchi, E. (1996). Spectral sensitivity of single photoreceptor cells in the eyes of the ctenid spider This research was supported in part by a Grant-in-Aid for Cupiennius salei Keys. Zool. Sci. 13, 199Ð202. Scientific Research from the Ministry of Education, Science Yamashita, S. (1985). Photoreceptor cells in the spider eye: spectral and Culture of Japan. sensitivity and efferent control. In Neurobiology of Arachnids (ed. F. G. Barth), pp. 103Ð117. Berlin, Heidelberg, New York: Springer Verlag. References Yamashita, S. and Tateda, H. (1978). Spectral sensitivities of the Barlow, R. B., Jr, Chamberlain, S. C. and Lehman, H. K. (1989). anterior median eyes of the orb web spiders, Argiope bruennichii Circadian rhythms in the invertebrate retina. In Facets of Vision and A. amoena. J. Exp. Biol. 74, 47Ð57. (ed. D. G. Stavenga and R. C. Hardie), pp. 257Ð280. Berlin, Yamashita, S. and Tateda, H. (1981). Efferent neural control in the Heidelberg, New York: Springer Verlag. eyes of orb weaving spiders. J. Comp. Physiol. 143, 477Ð483.