Spectral sensitivity studies on the whirligig beetle, Dineutes ciliatus

7. InsectF’hysid.,1967,Vol. 13,pp. 621 to 633. Pergamon Press Ltd.

Printed in Great Btitaik

SPECTRAL SENSITIVITY STUDIES ON THE WHIRLIGIG BEETLE, DINEUTES ClLIATUS RUTH R. BENNETT* Department of Biology, Tufts University, Medford, Massachusetts (Rectirzd 15 August 1966) Abstract-Spectral sensitivity curves of dorsal and ventral eyes of Dineutes ciliatus, using a constant electroretinogram response as an index of sensitivity, show a peak of sensitivity at 520 rnp and a U.V. ‘shoulder’. With long-wavelength light adaptation the 520 rnp peak becomes more depressed than the U.V. shoulder, and under these conditions the ventral eye has a far greater U.Y. sensitivity than the dorsal eye. Variations in waveform of electroretinograms throughout the spectrum, non-parallel response-energy functions, and the results of selective adaptation experiments indicate that both pairs of eyes probablypossess at least two visual pigments, one absorbing in the visible and one in the U.V. region of the spectrum. INTRODUCTION

RELATIVELYfew insects, representing six orders, have been subjected to detailed spectral sensitivity analysis. A variety of physiological approaches, including selective adaptation of an eye, have been used with four insects, representing four orders, in attempts to discover the number and characteristics of the visual pigments possessed by the insect (the fly: see summary by BURKHARDT,1964, and GOLDSMITH, 1965; the cockroach: WALTHER, 1958; the dragonfly: RUCK, 1965; the honeybee: see summary by GOLDSMITH,1964, and AUTRUM and VON ZWEHL, 1964). These four insects seem to have in common the following characteristics: (a) More than one visual pigment in the compound eye as a whole; and (b) physiologically ‘divided’ compound eyes, where a visual pigment is present in one area but not in another area of the eye. The Gyrinidae (whirligig beetles) have horizontally divided eyes-when a beetle swims on the surface of the water the ventral pair of eyes are completely submerged while the dorsal pair remain totally in an air environment. Morphological investigations (BOTT, 1928) and some physiological work (CARTHY and GOODMAN, 1964) on gyrinid eyes have revealed only slight differences between the two pairs of eyes. No spectral sensitivity studies on whirligig beetles have been reported. Differences in visual pigments, either in quantity or quality, might be expected if the uses to which the two pairs of eyes were put differed in a way which made it advantageous to have such differences, and/or if the spectral composition of the two environments caused selection for such differences. * Present address: University of Massachusetts, Boston. 621

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RUTH R. BENNETT

The present investigation of the spectral sensitivity of Dineutes ciliatus had the following purposes: (a) To discover whether the ventral and dorsal eyes of the whirligig beetle differed in spectral sensitivity, and (b) to discover, if possible, the number and characteristics of the visual pigments possessed by this beetle. MATERIALS

AND

METHODS

Optical apparatus: The optical stimulator is shown in Fig. 1. Test flashes of known wavelength, intensity, and duration could be delivered to the insect eye with this apparatus; in addition, the eye could be exposed to an adapting light (not shown in Fig. 1).

I

FIG. 1. Diagrammatic

Nepal

densiiy

filfers

sketch of optimal stimulator.

The test source was a tungsten projection bulb, operated through a voltage stabilizer. A Farrand u.v.-visible grating monochromator (O-5 mm slits, 5 rnp half-band width) was used in conjunction with Corning coloured filters to reduce stray light and a heat filter (aqueous 0.02 M CuCl, in a glass tank) to eliminate i.r. light. The intensity of the test beam was controlled with a calibrated compensated glass wedge and with calibrated Wratten neutral density filters. Wedge settings could be made without knowledge of their optical density; the latter could be read off after obtaining the desired response from an experimental animal. An RCA 917 phototube, calibrated by RCA for relative sensitivity throughout the spectrum, was used to determine the relative incident light intensity at the cornea from 380 rnp to 620 rnp, when no neutral density filters were in the light path. An approximation of the absolute energy incident at the cornea was made with a calibrated Eppley thermopile: threshold responses (20-30 ~LVERG height)

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from dorsal and ventral eyes were obtained when 105-10’ quanta/set of 520 rnp light were striking the cornea (illuminated eye surface estimated as being about O-2 mm2). Two adapting light systems were used; in both, the final coloured light was focused on one end of a fibre optic light pipe. The light pipe led into the chamber containing the beetle and was positioned so that the entire eye was illuminated. In one series of experiments light from a tungsten microscope lamp was focused into a collimating lens system; light then passed through a Corning coloured filter (see Table 1) and was focused on the end of the light pipe. In a second series of experiments a mercury lamp and a Bausch and Lomb monochromator were used to obtain monochromatic light (see Table 1). The intensity of each

TABLE

l--IPROPERTIES OF ADAPTINGLIGHTS

Adapting light

Filter or monochromator

Wavelength

‘Ultraviolet’ Blue Green Yellow Red

Coming filter 7-39 or 7-51 Monochromator Monochromator Monochromator Coming filter 2-62 or 2-73

<400 435 546 579 >570

(mcl>

adapting light was fixed at a value which gave, in preliminary experiments, a reasonable reduction in visual sensitivity. The actual intensity of adapting light at the eye may have varied slightly from experiment to experiment, due to small variations in the position of the light pipe relative to the eye. Animal preparation and recording methods : Whirligig beetles (Dineutes ciliatus Forsb.) were collected from streams in Concord and Harvard, Mass., maintained in an aquarium on a diet of freshly killed flies, and were usually tested within 2 weeks of capture. Intact, unanaesthetized beetles were mounted on a platform and their head and thorax immobilized with Tackiwax; the whole eye to be tested was exposed to the stimulating light through a small hole in an aluminium foil mask. Uninsulated, sharpened steel electrodes were used for recording electroretinograms. The active electrode pierced the cornea of the illuminated eye at the focus of the light beam and the indifferent electrode was positioned subcorneally in the other (shielded) eye of the pair under study. Recordings from the shielded eye with regard to an electrode in the abdomen showed no potential changes occurring in the shielded eye when light was stimulating the unshielded eye. In some experiments, a glass microelectrode (tip diameter approx. 1 p, resistance 2-3 MQ) was inserted into the illuminated eye through a small hole in the cornea, and was used in conjunction with the two steel electrodes. The electrodes were Ied into the input of a Grass P6 amplifier, used push-pull and direct-coupled or condenser-coupled; potentials were displayed on a

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RUTH R. BENNETT

Textronix 502 dual-beam oscilloscope and recorded on film. The oscilloscope was triggered by a Grass S4 stimulator which drove the electromagnetic shutter. In all records, negativity of the active electrode is indicated by an upward deflexion of the oscilloscope trace. Preliminary studies showed that both eyes recovered full sensitivity, following exposure to bright light, in 30-60 min. CARTHY and GOODMAN(1964) found this was also the case for Gyrinus bicolor. Prior to spectral sensitivity experiments, animals were dark adapted for at least 1 hr after positioning. Test flashes of 200 msec were delivered to the eye, starting with a low intensity, until the desired constant response was obtained (see below). Responses to test flashes of wavelengths from 380 rnp to 620 rnp were investigated and double checked. One of the adapting lights was then turned on and after 15 min light adaptation, the spectral sensitivity of the eye in the presence of the adapting light was determined. In the first series of experiments each eye was investigated with the ‘u.v.’ and red adapting lights; in the second series each eye was tested with the 3 mercuryline adapting lights. Each eye thus received a dark-adapted test plus two or three adapting light tests. Fifteen min were allowed for adaptation to each new adapting light, with no dark adaptation during this part of the experiment. Each beetle remained in the experimental situation for about 4 to 5 hr; it was then released and placed in an aquarium. Beetles behaved normally after experiments and remained alive for months afterwards. They were usually used for a second experiment on the other pair of eyes within a day or two of the first experiment. RESULTS General character of the electroretinogram In all dorsal eyes of Dinettes ciliatus, the waveform of the electroretinogram (ERG) is fairly simple under normal recording conditions. It consists of a comeanegative wave; the waveforms of dorsal eye ERGS can be matched at all wavelengths by adjustments of light intensity. The waveform of the ERG-from ventral DARK

BLUE

GREEN

YELLOW

FIG. 2. Ventral eye ERGS from Dinettes diutus, dark adapted l$ hr (first column) or light adapted to mercury-line adapting lights (see Table 1). Intensities at each test wavelength were adjusted to produce 250 PV initial negative on-waves (a.c. amplification). In two cases, not enough intensity was available from the optical stimulator to obtain the full 250 PV from this eye (green adapted, 380 and 620 mp); in some of the other cases shown the height is not exactly 250 r-;V, but was one of a series of responses which averaged 250 pV. Light flashes of 200 msec duration were used (bottom trace in each column).

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eyes is sometimes similar to that described above for dorsal eyes (e.g. Fig. 2, darkadapted column), but more often it is complex and contains a positive on-wave and a negative off-wave in addition to the negative on-wave. Light adaptation of ventral eyes can cause the appearance of these complexities (Fig. 2). Waveforms elicited by light flashes of different wavelengths cannot be matched by alterations in the intensity of light when the positive on-wave and negative off-wave are present in the ERG (Figs. 2, 3).

I

2

mV

4-L -If-

I

5 mV

“li* I 1

mV

200 msec

FIG. 3.

Ventral eye ERGS from Dine&es ciliatus; a.c. amplification. Eye dark adapted for 10 min. Intensity at each of two wavelengths was increased by about 0.5 log units in each frame. The 5 ERGS elicited by light of 380 rnp (first column) do not match the 9 ERGS elicited by light of 500 rnp (second and third columns) except at very low intensities. A new calibration mark is placed to the right of each ERG every time the amplification changes.

An attempt was made to clarify the source of the various potentials seen in the ERG, using a method similar to that of RUCK (1961). Three electrodes were used: the two conventional leads (E,, the active electrode; E,, the indifferent electrode) plus a microelectrode (E,) which could be passed through the receptor cell layer into the lamina ganglionaris. In ventral eyes which had complex ERGS under normal recording conditions (E1-Es) only simple, cornea-negative on-waves were apparent with leads El-Es, when E, was above the basement membrane (Fig. 4). Thus the positive on-wave and negative off-wave are probably generated by areas below the basement membrane in the ventral eye of D&z&s, as they are in the fly (RUCK, 1961) and in other insects (e.g. a butterfly: SWIHART, 1964). They wax and wane together with changes in the wavelength of the stimulus (see Figs. 2, 3), independently of the negative on-wave.

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RUTH R. BENNETT

FIG. 4. Ventral eye ERGS, 3-electrode experiment; d.c. amplification. E, was just beneath the cornea, El slightly deeper; both were well above the basement membrane. Millivolt scale applies to both records. 500 q light stimuli of 200 msec duration (arrows).

In the dorsal eye of Dine&es, positive on-waves and negative off-waves similar to those found in the ventral eye were seen when leads E,-E, were used, after E, had been advanced to the vicinity of the lamina ganglionaris (Fig. 5). It is not known why the dorsal eye never showed these complexities with conventional

FIG. 5. Dorsal eye ERGS, 3-electrode experiment; d.c. amplification. EB at a depth of 420~ in the eye (at or below the level of the lamina ganglionaris). Light stimulus 100 times less intense for top record (Er-ITa) than for lower 2 records. O-5mv scale placed at right of each record. 600 rnp light stimuli of 200 msec duration (arrows).

Fl¢;. 6. Dorsal (top a n d centre) a n d ventral (lower) eye sections of Dineute.~ ciliatus. B o u i n fixation, celloidin e m b e d d i n g , G i e m s a blood stain in top and lower sections, M a l l o r y ' s stain in c e n t r e section. Before fixation, I dorsal eye was light a d a p t e d while t h e o t h e r 3 were kept in darkness ; all 4 eyes showed similar p i g m e n t positions, c, cornea; cc, crystalline cones; rc, retinula cells; bin, b a s e m e n t m e m b r a n e ; ax, axons of r e t i n u l a cells; lg, lamina ganglionaris; me, medulla externa.

SPECTRAL SENSITIVITYSTUDIESONTHEwI-IIRLIGIGBEETLE

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leads (Z&-Q but an untested hypothesis is that the difference in the size and relative positions of the ganglia below the basement membrane in the dorsal and ventral eyes of gyrinids (Fig. 6 ; see also BOTT, 1928) plays a decisive r61e in ERG waveform difference. There is much evidence that the cornea-negative on-waves found in most insect ERGS arise from the activity of the receptor cells ; the results described above for Dinettes agree with the data on other insects. Postsynaptic potentials may add to or subtract from the negative on-wave in a yet undetermined amount, even when the ERG appears ‘simple’. Furthermore, the responses of different photoreceptor types may facilitate or inhibit one another. These considerations must be kept in mind when analysing the data given below. The ERG as a function ofintensity and wavelength of light. The relationship between the intensity of the stimulus and the height of the initial or sustained negative on-wave in dark-adapted dorsal and ventral eyes depends upon the wavelength of the stimulus. Response curves are not parallel ; those plotted for near-u.v. light have shallower slopes than those plotted for longer wavelengths. Two examples are shown in Fig. 7; a few more examples may be found in BENNETT (1965). Non-parallel response curves are a property of both eyes, using either d.c. or a.c. recording methods, and measuring either the initial or the sustained negative’on-wave of the ERG.

0.7 -

>E

DORSAL

0.5 -

0.11

I

1

L

I

1

2 LOG

,

3

I

INTENBlTY

FIG. 7. Relationship between the height of the sustained negative on-wave, measured from film records, and the intensity of monochromatic light in a dorsal and a ventral eye of two Dineutes ciliate; d.c. amplification used. The curves for different wavelengths have been moved arbitrary distances along the abscissa in order to show the differences in slopes more clearly.

I

4

628

RUTHR.BENNETT

Spectral sensitivity studies. The average relative spectral sensitivity of 10 darkadapted dorsal eyes and 6 dark-adapted ventral eyes is shown in Fig. 8. The constant response criterion for each eye was a 250 PV initial negative on-response (solid lines) and the ERG waveform was ‘simple’ in all of these eyes. The average spectral sensitivity of 4 additional ventral eyes, where the constant response criterion was a 250 ~LVsustained negative wave (these were eyes which produced r 100 -

IO-

5-

\ 2"""11""L 380

440

500 Wavelength,

560

62

m,u

FIG. 8. Spectral sensitivity curves of dark-adapted eyes of Din&es ciliatzks. The reciprocal of the number of quanta needed to elicit a 250 FV initial negative on-wave to flashes of 200 msec is plotted along the ordinate (relative sensitivity) with the sensitivity at 520 rnp made equal to 100 before averaging. Ten dorsal eye experiments and 6 ventral eye experiments are averaged with standard deviations (vertical lines) indicated. Dashed line: Average of 4 ventral eye experiments where the criterion response was a 250 ~LV sustained negative on-response in a complex ERG. Between 520 rnp and 620 rnp the points for this curve coincided closely with those for the solid line and are therefore not shown. Note semi-log nature of this graph and that of Fig. 9.

SPECTRAL SENSITIVITY

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complex ERGS), is shown by the dashed line in Fig. 8. In both eyes there is a major peak of sensitivity at 520 rnp; both show a shoulder of sensitivity in the near U.V. Sensitivity at wavelengths shorter than 520 rnp is enhanced relative to

\

VENTRAL

460

420

500

540

580 WAVELENGTH.

FIG.

9.

Spectral

Dineutes ciliatus. values

except

closely

with

sensitivity Sensitivity

curves

of

mp

light-adapted

of light-adapted

in the case of the green-adapted that

lower

position

eyes

represents

of yellow-adapted

on the ordinate an

average

eyes

for purposes

and

dorsal has been

of clarity.

of 3 to 6 experiments; eyes are those of Fig.

4I

and

eyes shown

8.

dark-adapted

relative

eyes

;

this curve

arbitrarily

eyes

of

to dark-adapted coincided

displaced

to a

Each curve for light-adapted the curves for dark-adapted

630

RUTHR.BENNETT

longer wavelengths in the ventral eye when the ‘response criterion is the sustained negative wave of a’complex ERG; when other constant response criteria are chosen, such as the positive on-wave or negative off-wave of a complex ERG, there is some additional enhancement of the U.V. shoulder (see BENNETT, 1965). The ventral eye is somewhat more sensitive than the dorsal eye at wavelengths shorter than 420 rnp (solid lines, Fig. S), but otherwise the two eyes are similar in the dark-adapted state. The average absolute sensitivities of the two eye are similar at 520 mp. The ventral eye proved to have a far higher U.V. sensitivity under conditions of selective light adaptation than did the dorsal eye (Fig. 9). While short wavelength light adaptation (blue and U.V. curves, Fig. 9) decreased the sensitivity of both eyes almost uniformly throughout the spectrum, adaptation with longer wavelengths (green, yellow, and red curves) decreased the sensitivity of,both eyes less drastically in the near U.V. than in the visible part of the spectrum. The effect of green- and yellow-adapting lights on the ventral eye is particularly striking. For instance, the average sensitivity of 4 yellow-adapted ventral eyes at 380 rnp was forty times that at 520 mp. The curves of Figs. 8 and 9 do not show anything definitive regarding the number of visible-absorbing pigments present in whirligig beetle eyes. Absorption curves of a mixture of two pigments (where the peaks are close together) are similar enough to the absorption curve of a single pigment with an intermediate peak, so that the present method might never distinguish between the several alternatives regarding the number of visual pigments absorbing in the visible region of the spectrum. It is interesting to note, however, that the average of 10 dark-adapted dorsal eyes shows a considerably higher standard deviation than does the average of 6 dark-adapted ventral eyes at wavelengths longer than 520 mp. The curves from light-adapted dorsal eyes are quite ‘bumpy’ in the visible region of the spectrum, compared to the fairly smooth curves from ventral eyes. Care was taken in positioning the eyes for electrode placement, the same region of each eye was always used, the methodology was the same for both eyes, and experiments were alternated between the two eyes. The greater variability of the dorsal eye thus appears to be real and may reflect the presence of more than one visibleabsorbing pigment in that eye. DISCUSSION

The results described above-ERG waveform differences, selective adaptation results, and non-parallel response-energy functions-cannot all be easily accounted for unless there are at least two visual pigments in each eye, one absorbing in the U.V. region of the spectrum and one in the visible region. The following facts may be examined: (1) ERG waveforms from the ventral eye which contain contributions from postsynaptic areas cannot be ‘matched’ throughout the spectrum. Often there are two transitions in waveform; the near-u.v. and red ends of the spectrum elicit more postsynaptic contributions, relative to receptor contributions, than does the

SPECTRAL SENSITIVITY

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region around 520 mp. GOLDSMITH(1965) has shown that ERG waveform in the fly eye can depend on the number of receptors stimulated; larger numbers of receptors stimulated (e.g. through a shielding pigment) lead to larger postsynaptic contributions, relative to receptor contributions, in the fly ERG. If the black shielding pigment in the whirligig beetle eye were more transparent to near-u.v. and red light than to the middle region of the spectrum, the inability to match waveforms could be due to the shielding pigment. Alternatively, there might be more than one visual pigment in separate receptors, with appropriate higher-order connexions, which would bring on different amounts of postsynaptic activity in different areas of the spectrum. (2) Both dorsal and ventral eyes have a spectral sensitivity which can be altered by long wavelength light adaptation; the latter depresses the major peak at 520 rnp more than the near-u.v. shoulder. The red peak of sensitivity in the fly eye can be eliminated by red-light adaptation or enhanced by blue-light adaptation, and it is probable that this is due to a selective transmission of red light by the red shielding pigment rather than a ‘red’ receptor (GOLDSMITH,1965). The results of selective adaptation in the whirligig beetle are similar to those on the honeybee and the cockroach, and all of these insects have black shielding pigments which may be more transparent in the U.V. than in the visible spectrum. The bee, however, does have receptors specifically sensitive to U.V. light alone (AUTRUMand VON ZWEHL, 1964). In the whirligig beetle, the structure of the two eyes above the basement membrane is similar and the shielding pigments look similar in colour and distribution, but the eyes are different after selective adaptation with long wavelength radiation in their U.V. sensitivity. (3) Both dorsal and ventral eyes exhibit the phenomenon of non-parallel response-energy curves. Receptor responses to near-u.v. radiations increase more slowly as a function of light intensity than do the responses to longer wavelengths. If certain assumptions are made (see GOLDSMITH, 1965), the response-energy function should be steeper for regions of the spectrum where shielding pigments transmit light, shallower for regions where shielding pigments absorb light. The results on red- and white-eyed flies appear to bear out this hypothesis (GOLDSMITH, 1965). If the black shielding pigments in bees, cockroaches, and whirligig beetles actually do transmit more light in the near-u.v. than in the visible region of the spectrum (see point 2, above), response-energy curves should be steeper in the near U.V. In the bee they are parallel (GOLDSMITH, 1960) and in the whirligig beetle they are shallower in this region. Another insect with black eyes is Carabus nemoralis ; if the spectral sensitivity curves found for this beetle are examined it is apparent that response curves, if plotted, would be shallower at wavelengths below 480 rnp than at wavelengths above 480 rnp (see Fig. 5a of HASSELMANN,1962). ‘Black’ shielding pigments could cause these non-parallel response-energy functions by transmitting more light at long wavelengths. In the whirligig beetle, however, preliminary microspectrophotometric studies have shown that the pigment granules found in the eyes have a high absorption throughout the green region of the spectrum, where the response-energy function is usually as steep as it is in the’red region

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RUTH R. BENNETT

of the spectrum. If non-parallel response curves are not a function of shielding pigments, a number of alternatives are possible, and all of these involve the functioning of at least two different visual pigments in the eye. It is clear that behavioural studies are needed before certain questions, raised by this study, can be answered. For instance, why is the ventral eye more sensitive than the dorsal eye to U.V. light (and strikingly so under conditions of long wavelength light adaptation) ? The best transmission of light by distilled water is of wavelengths between 400 and 560 rnp; at 360 rnp as much as 20 per cent/m is absorbed (see JA~MESand BIRGE, 1938; CLARKE, 1939). An untested possibility which would explain the difference in the two eyes is that the ventral eye represents an adaptation to an underwater environment. Here, if it is advantageous to see in the u.v., sensitivity must be increased relative to an eye adapted to an air environment in order to compensate for the reduced amount of U.V. light present. However, physiological spectral sensitivity studies do not necessarily reflect the behavioural spectral sensitivity of an animal (e.g. the preference of bees for U.V. light would not be predicted from physiological spectral sensitivity measurements-see GOLDSMITH, 1960); thus one cannot be certain that the two eyes of the whirligig beetle differ for the animal in the same way that they appear to differ to the physiologist studying the eyes. Another unanswered question could also be asked of a number of other insects: if the whirligig beetle does possess at least two visual pigments in each eye, do these pigments serve only to broaden the spectral range of the insect, or are they used for ‘colour vision’ -that is, can these insects discriminate between two different wavelengths of light ? If so, of what advantage is it for these insects to possess this ability ? Behavioural studies on insects will, hopefully, answer these and other questions concerning insect vision. Meanwhile, physiological studies such as the present one can help to define the areas where behavioural work is needed. Acknowledgements-The work was supported in part by Training Grant Al-T2-32 from the National Institutes of Health to Tufts University, and in part during the tenure of a Title 4 Graduate Fellowship. I wish to thank Dr. PHILIP RUCICfor his help, advice, and critical evaluations during the course of these studies. REFERENCES

A~JTRUMH. and VAN ZWEHLV. (1964) Die spektrale Empfindlicbkeit einzelner Sehzellen

des Bienenauges. Z. erergl. Physiol. 48, 357-384. BENNETT R. (1965) Vision in the whirligig beetle, Dineutes ciliatus Forsb. Ph.D. Thesis, Tufts University, Medford, Mass. BOTT H. R. (1928) Beitrage zur Kenntnis von Gyrinus natutm substriutus Steph.-I. Lebensweise und Entwicklung. II. Der Sehapparat. Z. Morph. bkol. Tiere 10,207-306. BURKHARDT D. (1964) Colour discrimination in insects. In Adv. Insect Physiol. 2, 131-174. CARTHY J. D. and GOODMANL. J. (1964) An electrophysiological investigation of the divided eye of, Gyrinus bicolm F. -7. Insect Physiol. 10,431-436. CLARKE G. L. (1939) Utilization of solar energy by aquatic organisms. Problems in Lake Biology. Am. Ass. Adv. Sci. Publ. 10, pp. 27-38.

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GOLDSMITHT. H. (1960) The nature of the retinal action potential and the spectral sensitivities of ultraviolet and green receptor systems of the compound eye of the worker honeybee. J. gen. Physiol. 43, 775-799. GOLDSMITHT. H. (1964) The visual system of insects. In The Physiology of Ikecta (Ed. by ROCKXEIN M.) 1,397-462.Academic Press, New York. GOLDSMITHT. H. (1965) Do flies have a red receptor ? J. gen. Physiol. 49, 265-287. HASSELMANNE.-M. (1962) l?ber die relative spectrale Empfmdlichkeit von Ktier- und Schmetterlingsaugen bei verschiedenen Helligkeiten. Zool. Jb. (Physiol.) 69, 537-576. JAMESH. R. and BIRGE E. E. (1938) A laboratory study of the absorption of light by lake waters. Trans. Wis. Acad. Sci. Arts Lett. 31, 1-154. RUCK P. (1961) Photoreceptor cell response and flicker fusion frequency in the compound eye of the fly, Lucilia sericata (Meigen). Biol. Bull., Woods Hole 120,375-383. RUCK P. (1965) The components of the visual system of a dragonfly. J. gen. Physiol. 49, 289-307. SWIHARTS. L. (1964) The nature of the electroretinogram of a tropical butterfly. 3. Insect Physiol. 10,547-562. WALTHER J. B. (1958) Changes induced in spectral sensitivity and form of retinal action potential of the cockroach eye by selective adaptation. J. lizsect Physiol. 2, 142-151.