Properties of pupil mechanisms in owl-fly Ascalaphus macaronius (Neuroptera)

Regulation of light flux by pupil mechanisms in the UV-sensitive superposition eye of owl-fly Ascalaphus macaronius (Neuroptera) was studied with a fast reflection microspectrophotometric technique. The spectral sensitivity of pupil reaction, which was calculated on the basis of changes of transient amplitude reflection, was almost identical with the one of Deilephila eye. This indicates that in spite of different life styles and spectral sensitivities of photoreceptors, pupil closing is triggered by the same photosensitive structure in both eyes. By measuring the spectra of reflected light from the Ascalaphus eye between 400 and 700 nm after different dark periods following light stimulation, it was established that the restoration of reflection was much faster in the red than in the blue spectral range. Based on this, we propose that two different pupil mechanisms with different spectral absorption characteristics are involved in light-flux regulation. Fast-reacting pupil is probably represented by screening pigment migration in the secondary pigment cells and a slow blue-absorbing system by the activity in primary pigment cells. The importance of two different pupils for the photoregeneration of visual pigment is discussed. Accepted: 1 October 1998     

Le contrôle automatique du flux lumineux dans l'oeil composé des Diptères

In the compound eye of the fly Musca, tiny pigment granules move within the cytoplasm of receptor cells Nos. 1–6 and cluster along the wall of the rhabdomeres under light adaptation, thus attenuating the light flux to which the visual pigment is exposed (Kirschfeld and Franceschini, 1969). Two recently developed optical methods (the neutralization of the cornea and the deep pseudopupil) combined with antidromic and orthodromic illumination of the eye (Fig. 1) make it possible to analyse the properties of the mechanism at the level of the single cell, in live and intact insects (Drosophila and Musca). The mechanism is shown to be an efficient attenuator in the spectral range (blue-green) where cells Nos. 1–6 have been reported to be maximally sensitive (Figs. 4c and d, 5b and 11b). In spite of the fact that the granules do not penetrate into the rhabdomere, the attenuation spectrum they bring about closely matches the absorption spectrum of the substance of which they are composed (ommochrome pigment, dotted curve in Fig. 11b). The dramatic increase in reflectance of the receptors after light adaptation (Figs. 3, 4b, 5a and 11a) can be explained as a mere by-product of the high absorption index of the ommochrome pigment, especially if one takes into account the phenomenon of anomalous dispersion (Chapter 8). The vivid green or yellow colour of the rhabdomeres would thus have a physical origin comparable to a metallic glint. Contrasting with the lens eye in which the pupillary mechanism is a common attenuator for both receptor types (rods and cones), the compound eye of higher Diptera is equiped with two types of pupils adapted respectively to both visual subsystems. A scotopic pupil is present in each of the six cells (Nos. 1–6) whose signals are gathered in a common cartridge of the first optic ganglion. This pupil comes into play at a moderate luminance (0,3 cd/m² in Drosophila; 3 to 10 cd/m² in Musca. Figs 13, 14, 15, 16). A photopic pupil is present in the central cell No. 7 whose signal reaches one column of the second optic ganglion. Attenuating the light flux for both central cells 7 and 8, the photopic pupil has its threshold about two decades higher than the scotopic pupil, just at the point where the latter reaches saturation (Fig. 3b, e-State II of Figs. 6b and 15). The photopic pupil itself saturates at a luminance one to two decades higher still (Fig. 3c, f=State III of Figs. 6c and 15). The two-decades-shift in threshold of these pupil-mechanisms supports the view that receptors 1–6 are a scotopic subsystem, receptors 7 and 8 a photopic subsystem of the dipteran eye. The luminance-threshold of the scotopic pupil (as determined with the apparatus described in Fig. 2) appears to be located at least 3.5 decades (Drosophila) or even 5 decades (Musca) higher than the absolute threshold of movement perception (Fig. 16). After a long period (1 hr) of darkness a light step of high intensity can close the scotopic pupil within about 10 sec (time constant 2 sec as in Fig. 9) and the photopic pupil within no less than 30–60 sec. Some mutants of Drosophila possess only a scotopic pupil (w , Figs. 4 and 5) whereas ommochrome deficient mutants lack both types of pupil (v, cn, see Fig. 7c, d). Comparable reflectance changes, accomplished within about 60 sec of light adaptation, are described for two insects having fused rhabdomes: the bee and the locust (Fig. 17).     

The responses of the pupil of Gekko gekko to external light stimulus

1. The responses of the pupil of a nocturnal gecko (Gekko gekko) to external light stimulus were studied. 2. The responses of the pupil are determined by light entering the pupil and not by light acting directly on the iris. 3. The responses of the pupil are very uniform in sensitivity including spectral sensitivity for light coming in different directions to the eye. 4. The possible change in area of the pupil is more than 300-fold and probably represents an effort to shield the pure rod retina from saturating light intensities. 5. The pupil continues to contract sharply for changes in external light intensity which give retinal illuminations corresponding to 10⁶ quanta/sec. striking a retinal rod. 6. There is a large degree of spatial summation of the response; circular external light fields subtending 5 and 140° giving the same illumination at the pupil give approximately the same pupil response. 7. The spectral sensitivity curve agrees with the absorption curve of an extracted pigment from a closely related gecko described by Crescitelli in the followig paper. It is similar to the human scotopic curve but its maximum is displaced about 20 to 30 mµ towards the red end of the spectrum. The fall in sensitivity towards the red end of the spectrum is described by the equation See PDF for Equation     

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

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 graminis. 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.     

Direct Action of Light in Naturally Pigmented Muscle Fibers : I. Action spectrum for contraction in eel iris sphincter

Contraction due to light in excised eel irises appears to follow a simple first order law. The action spectrum for contraction has a maximum which agrees with the eel rhodopsin absorption maximum. Inasmuch as rhodopsin is the rod pigment-opsin complex and the iris sphincter pupillae evolves from the pigment epithelium of the retina in the region of the iris, the muscle pigment might be the same as the visual pigment. In the human eye the contraction of the iris sphincter is activated only by light incident on the retina and the pupil diameter varies inversely with the square root of the light intensity. The inverse first power relation observed in the present experiments suggests a more primitive origin for the light reaction in eel irises. Relaxation is a much slower process and can be approximated as the sum of two first order processes.     

Screening pigment migration in a sphingid moth is triggered by light near the cornea

Summary It is possible to project a horizontal line of light onto a moth's cornea and simultaneously a vertical line onto the retina (Fig. 1). In the dark adapted eye of a sphingid moth (Theretra latreilli) this resulted in the appearance of a horizontal line of pigment across the eye (Fig. 2). This proves that the trigger for pigment migration is near the cornea, presumably in the pigment cells themselves, and not in the receptors.     

Angular and spectral sensitivity of fly photoreceptors. III. Dependence on the pupil mechanism in the blowfly<Emphasis Type="Italic"> Calliphora</Emphasis>

A wave optics model for the facet lens-rhabdomere system of fly eyes is used to analyze the dependence of the angular and spectral sensitivity of R1–6 photoreceptors on the pupil mechanism. This assembly of light-absorbing pigment granules in the soma interacts with the waveguide modes propagating in the rhabdomere. A fly rhabdomere carries two modes in the middle wavelength range and four modes at short wavelengths, depending on the rhabdomere diameter and the angle of the incident light flux. The extension of the mode to outside the rhabdomere strongly depends on wavelength, and this dependence plays a determinant role in the light control function of the pupil. The absorbance spectrum of the pigment in the pupil granules is severely depressed at short wavelengths by waveguide effects, resulting in a distinct blue peak. Accordingly, pupil closure suppresses the photoreceptors spectral sensitivity much more in the blue-green than in the UV. The pupil only narrows the angular sensitivity at short wavelengths. The geometrical size of the rhabdomere governs the angular sensitivity of fly photoreceptors in the dark-adapted state, but diffraction takes over in the fully light-adapted state.     

Do retinula cells trigger the screening pigment migration in the eye of the mothEphestia kuehniella?

Summary Simultaneous recordings of reflectance and the electroretinogram (ERG) of the meal moth superposition eye show a good match between the action spectrum of screening pigment migration and the spectral sensitivity curve (Fig. 5). These spectra correspond with the absorption spectrum of a xanthopsin X530 that has been evidenced in the eye. No correlation was found with the extinction spectrum of the pigment granules themselves (Fig. 7). The results suggest, that the photomechanical reaction is controlled by the visual pigment.     

Adaptation of a deep-sea cephalopod to the photic environment. Evidence for three visual pigments

Watasenia scintillans, a bioluminescent deep-sea squid, has a specially developed eye with a large open pupil and three visual pigments. Photoreceptor cells (outer segment: 476 micron; inner segment: 99 micron) were long in the small area of the ventral retina receiving downwelling light, whereas they were short (outer segment: 207 micron; inner segment: 44 micron) in the other regions of the retina. The short photoreceptor cells contained the visual pigment with retinal (lambda max approximately 484 nm), probably for the purpose of adapting to their environmental light. The outer segment of the long photoreceptor cells consisted of two strata, a pinkish proximal area and a yellow distal area. The visual pigment with 3-dehydroretinal (lambda max approximately 500 nm) was located in the pinkish proximal area, giving high sensitivity at longer wavelengths. A newly found pigment (lambda max approximately 471 nm) was in the yellow distal area. The small area of the ventral retina containing two visual pigments is thought to have a high and broad spectral sensitivity, which is useful for distinguishing the bioluminescence of squids of the same species in their environmental downwelling light. These findings were obtained by partial bleaching of the extracted pigment from various areas of the retina and by high-performance liquid chromatographic analysis of the chromophore, complemented by microscopic observations.     

Did neural pooling for night vision lead to the evolution of neural superposition eyes?

Observations of the infrared deep pseudopupil, optical determinations of the corneal nodal point, and histological methods were used to relate the visual fields of individual rhabdomeres to the array of ommatidial optical axes in four insects with open rhabdoms: the tenebrionid beetle Zophobas morio, the earwig Forficula auricularia, the crane fly Tipula pruinosa, and the backswimmer Notonecta glauca.The open rhabdoms of all four species have a central pair of rhabdomeres surrounded by six peripheral rhabdomeres. At night, a distal pigment aperture is fully open and the rhabdom receives light over an angle approximately six times the interommatidial angle. Different rhabdomeres within the same ommatidium do not share the same visual axis, and the visual fields of the peripheral rhabdomeres overlap the optical axes of several near-by ommatidia. During the day, the pigment aperture is considerably smaller, and all rhabdomeres share the same visual field of about two interommatidial angles, or less, depending on the degree of light adaptation. The pigment aperture serves two functions: (1) it allows the circadian rhythm to switch between the night and day sampling patterns, and (2) it works as a light driven pupil during the day.Theoretical considerations suggest that, in the night eye, the peripheral retinula cells are involved in neural pooling in the lamina, with asymmetric pooling fields matching the visual fields of the rhabdomeres. Such a system provides high sensitivity for nocturnal vision, and the open rhabdom has the potential of feeding information into parallel spatial channels with different tradeoffs between resolution and sensitivity. Modification of this operational principle to suit a strictly diurnal life, makes the contractile pigment aperture superfluous, and decreasing angular sensitivities together with decreasing pooling fields lead to a neural superposition eye.Abbreviations DPP deep pseudopupil - LMC large monopolar cell     

Retinal receptor types inAglais urticae andPieris brassicae (Lepidoptera), revealed by analysis of the electroretinogram obtained with Fourier interferometric stimulation (FIS)

Summary Electroretinograms obtained in the butterfliesAglais urticae andPieris brassicae by the procedure of Fourier interferometric stimulation (FIS) were used to construct spectral sensitivity curves. These curves, representing the combined responses of several receptor types, were approximated by summation of spectral sensitivity curves for individual pigments, and the presence of these pigments was corroborated by chromatic adaptation experiments. The results show that the retina in the compound eye ofAglais urticae contains 3 photopigments, with maximal absorption at ca. 360 nm, 460 nm and 530 nm, respectively (Fig. 5). The retina in the compound eye ofPieris brassicae has two subdivisions. In the dorsal region of the eye 3 photopigments were found, with maxima at ca. 360 nm, 450 nm and 560 nm (Fig. 8). In the medioventral region pigments with essentially the same maxima are present together with an additional, fourth long-wavelength component with effective maximal absorption at ca. 620 nm (Fig. 11). Its absorption curve is considerably narrower than would be expected for a rhodopsin with the same absorption maximum, and presumably results from the spectral combination of a photopigment and a photostable screening pigment.Abbreviations FIS Fourier interferometric stimulation - WLP White-light position - ERG Electroretinogram     

Colour in the eyes of insects

Many insect species have darkly coloured eyes, but distinct colours or patterns are frequently featured. A number of exemplary cases of flies and butterflies are discussed to illustrate our present knowledge of the physical basis of eye colours, their functional background, and the implications for insect colour vision. The screening pigments in the pigment cells commonly determine the eye colour. The red screening pigments of fly eyes and the dorsal eye regions of dragonflies allow stray light to photochemically restore photoconverted visual pigments. A similar role is played by yellow pigment granules inside the photoreceptor cells which function as a light-controlling pupil. Most insect eyes contain black screening pigments which prevent stray light to produce background noise in the photoreceptors. The eyes of tabanid flies are marked by strong metallic colours, due to multilayers in the corneal facet lenses. The corneal multilayers in the gold-green eyes of the deer fly Chrysops relictus reduce the lens transmission in the orange-green, thus narrowing the sensitivity spectrum of photoreceptors having a green absorbing rhodopsin. The tapetum in the eyes of butterflies probably enhances the spectral sensitivity of proximal long-wavelength photoreceptors. Pigment granules lining the rhabdom fine-tune the sensitivity spectra.     

Insect pupil mechanisms

Summary The hypothesis that the glow observable in dark adapted butterfly eyes is extinguished upon light adaptation by the action of migrating retinula cell pigment granules (Stavenga, 1975a) has been investigated. Experimental procedures applying optical methods to intact, living animals were similar to those used previously to investigate the migration of retinula cell pigment granules in Hymenoptera (Stavenga and Kuiper, 1977). The data obtained from nymphalid butterflies and Hymenoptera show close parallels, favouring the pigment migration hypothesis.The retinula cell pigment granules control the light flux in the butterfly rhabdom and hence are part of a pupil mechanism. The range of action of this pupil mechanism is about 3 log units of light intensity. The speed of pupil closure is slowed down with longer dark adaptation times. The way in which pupil processes can be distinguished from photochemical processes of the visual pigment is discussed.     

Daily changes of structure,function and rhodopsin content in the compound eye of the crabHemigrapsus sanguineus

The compound eye of the crab hemigrapsus sanguineus undergoes daily changes in morphology as determined by light and electron microscopy, both in the quantity of chromophore substances studied by HPLC and in visual sensitivity as shown by electrophysiological techniques. 1. At a temperature of 20 degrees C, the rhabdom occupation ratio (ROR) of an ommatidial retinula was 11.6% (maximum) at midnight, 8.0 times larger than the minimum value at midday (1.4%). 2. Observations by freeze-fracture revealed that the densities of intra-membranous particles (9-11 nm in diameter) of rhabdomeric membrane were ca. 2000/microns 2 and ca. 3000/microns 2 for night and daytime compound eyes, respectively. 3. Screening pigment granules migrated longitudinally and aggregated at night, but dispersed during the day. Reflecting pigment granules migrate transversally in the proximal half of the reticula layer i.e. cytoplasmic extensions containing reflecting pigment granules squeeze between neighbouring retinula cells causing optical isolation (Fig. 4). Thus the screening pigment granules within the retinula cells show longitudinal migration and radial movement so that the daytime rhabdoms are closely surrounded by the pigment granules. 4. At 20 degrees C, the total amount of chromophore of the visual pigment (11-cis and all-trans-retinal) was 1.4 times larger at night than during the day i.e. 46.6 pmol/eye at midnight and 33.2 pmol/eye at midday. Calculations of the total surface area of rhabdomeric membrane, total number of intra-membranous particles in rhabdomeric membrane and the total number of chromophore molecules in a compound eye, indicate that a considerable amount of chromophore-protein complex exists outside the rhabdom during the day. 5. The change in rhabdom size and quantity of chromophore were highly dependent on temperature. At 10 degrees C both rhabdom size and amount of chromophore stayed close to daytime levels throughout the 24 hours. 6. The intracellularly determined relative sensitivity of the dark adapted night eye to a point source of light was about twice as high as the dark-adapted day eye. Most of the increase in the sensitivity is attributed primarily to the effect of reflecting pigment migration around the basement membrane and, secondarily, to the changes in the amount and properties of the photoreceptive membrane. The results form the basis of a detailed discussion as to how an apposition eye can function possibly as a night-eye.     

Spectral sensitivities of retinular cells measured in intact,living flies by an optical method

Summary The spectral sensitivity of the peripheral retinular cells R1–6 in nine species of intact flies was determined using non-invasive, optical measurements of the increase in reflectance that accompanies the pupillary response. Our technique is to chronically illuminate a localized region of the eye with a long wavelength beam, adjusted to bring pupillary scattering above threshold, then, after stabilization, to stimulate with monochromatic flashes. A criterion increase in scattering is achieved at each wavelength by adjusting flash intensity. Univariance of the pupillary response is demonstrated by Fig. 3.Action spectra measured with this optical method are essentially the same as the published spectral sensitivity functions measured with intracellular electrophysiological methods (Fig. 4 forCalliphora, Fig. 5 forDrosophila, Fig. 7 forEristalis, and Fig. 8 forMusca). This holds for both the long wavelength peak and the high sensitivity in the UV as was consistently found in all investigated fly species.Spectral sensitivity functions for R1–6 of hover flies (family Syrphidae) are quite different in different regions of the same eye. There can also be substantial differences between the two sexes of the same species. The ventral pole of the eye of femaleAllograpta (Fig. 10) contains receptors with a major peak at 450 nm, similar to those ofEristalis. However, the dorsal pole of the same eye contains receptors with a major peak at 495 nm, similar to those ofCalliphom. Both dorsal and ventral regions of the maleToxomerus eye, and the ventral region of the female eye, contain only the 450 nm type of R1-6 (see Fig. 12). However, the dorsal region of the female eye also contains another spectral type of receptor that is maximally sensitive at long wavelength. Eyes of both sexes ofAllograpta (Figs. 10 and 11) contain a mixture of spectral types of receptors R1-6.We thank Dr. Chris Maier of the Connecticut Agricultural Experiment Station, for determination of the Syrphidae. This work was supported by grants EY01140 and EY00785 from the National Eye Institute, U.S.P.H.S., (to GDB), by the Connecticut Lions Eye Research Foundation (to GDB), and by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.), (to DGS).     

Strategies for retinal design in arthropod eyes of low F-number

Summary The superposition eyes and simple eyes of many arthropods have apertures (A) with a diameter bigger than, or about the same size as, the focal length of the eye (f). That is, these eyes have low F-numbers (f/A). Many of the light rays focussed onto a photoreceptor will not be trapped by total internal reflection in the photoreceptor and will therefore pass through and be absorbed in photoreceptors other than that for which the light was intended. This spread of light in the retina leads to a broadening of the angular-sensitivity function and a consequent degrading of the image at the retinal level. A number of solutions to this problem are found in nature, with the most effective that of isolating the photoreceptors with a sheath of either light-absorbing pigment or reflecting tapetum. A ray-tracing model was used to assess the relative merits of the tapetal and pigment sheath designs in low F-number superposition eyes, and also to investigate the effect of changing the refractive index and absorption coefficient of the rhabdom. Which sheathing solution is best depends on the quality of the image on the retina, on the spacing of the rhabdoms in the retina and on the intensity of light normally experienced by the eye. In a retina with closely packed rhabdoms, the model predicts full sheathing to be the optimal solution if the image is well-focussed, partial sheathing if poorly focussed and no sheathing if moderately well-focussed. In a retina with rhabdoms spaced apart and a well-focussed image, the model predicts partial sheathing to be optimal. A pigment sheath is predicted to be useful in eyes which experience bright light and have no need for high sensitivity. A tapetal sheath is predicted to be useful at any intensity. A survey of arthropod eyes with low F-number supports the predictions of the model.     

Effects of energy deprivation on the fly pupil mechanism: evidence for a rigor state

The energy dependence of the pupil pigment-migrations in the fly Musca domestica was studied in live animals, using optical techniques and nitrogen-gas induced anoxia. The results obtained can be summarized in 3 points:

1. Energy deficiency can make the pupil mechanism stop in any state, extreme or intermediate.
2. Anoxia induced during intermittent stimulation makes the pupil stop in the closed state (aggregated pigment granules).
3. During long-term anoxia the pupil very slowly opens (dispersal of pigment granules), irrespective of ambient intensity.

The slow anoxic opening (point 3) is more than 1000 times slower than that predicted for free diffusion of pigment granules in water. Assuming realistic values of cytoplasm viscosity, this implies that anoxia causes the pigment granules to attach to rigid structures in the cells, in analogy with the rigor state in anoxic muscles. The rigor phenomenon in the pupil mechanism prevents experimental discrimination between active and passive processes of pigment migration. Normal pupil opening has a time course which agrees reasonably with a passive diffusion process, but it is argued that an active transportation of granules away from the rhabdom is more likely in the dark adapted eye.     

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface (''rough eye'' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.¹). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. ²). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells (''hexagon'' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. ³).The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready⁴. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined^5,6.