Pupil Mechanism Research Articles

Automated Charting of the Visual Space of Housefly Compound Eyes.

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands of visual units called ommatidia. Each ommatidium samples the optical information from a small solid angle, with an approximate Gaussian-distributed sensitivity (half-width on the order of 1˚) centered around a visual axis. Together, the ommatidia gather the visual information from a nearly panoramic field of view. The spatial distribution of the visual axes thus determines the eye's spatial resolution. Knowledge of the optical organization of a compound eye and its visual acuity is crucial for quantitative studies of neural processing of the visual information. Here we present an automated procedure for mapping a compound eye's visual axes, using an intrinsic, in vivo optical phenomenon, the pseudopupil, and the pupil mechanism of the photoreceptor cells. We outline the optomechanical setup for scanning insect eyes and use experimental results obtained from a housefly, Musca domestica, to illustrate the steps in the measurement procedure.

The ocelli of Archaeognatha (Hexapoda): functional morphology, pigment migration and chemical nature of the reflective tapetum.

The ocelli of Archaeognatha, or jumping bristletails, differ from typical insect ocelli in shape and field of view. Although the shape of the lateral ocelli is highly variable among species, most Machiloidea have sole-shaped lateral ocelli beneath the compound eyes and a median ocellus that is oriented downward. This study investigated morphological and physiological aspects of the ocelli of Machilis hrabei and Lepismachilis spp. The light-reflecting ocellar tapetum in M. hrabei is made up of xanthine nanocrystals, as demonstrated by confocal Raman spectroscopy. Pigment granules in the photoreceptor cells move behind the tapetum in the dark-adapted state. Such a vertical pigment migration in combination with a tapetum has not been described for any insect ocellus so far. The pigment migration has a dynamic range of approximately 4 log units and is maximally sensitive to green light. Adaptation from darkness to bright light lasts over an hour, which is slow compared with the radial pupil mechanism in some dragonflies and locusts.

Visual Reliability and Information Rate in the Retina of a Nocturnal Bee

Nocturnal animals relying on vision typically have eyes that are optically and morphologically adapted for both increased sensitivity and greater information capacity in dim light. Here, we investigate whether adaptations for increased sensitivity also are found in their photoreceptors by using closely related and fast-flying nocturnal and diurnal bees as model animals. The nocturnal bee Megalopta genalis is capable of foraging and homing by using visually discriminated landmarks at starlight intensities. Megalopta's near relative, Lasioglossum leucozonium, performs these tasks only in bright sunshine. By recording intracellular responses to Gaussian white-noise stimuli, we show that photoreceptors in Megalopta actually code less information at most light levels than those in Lasioglossum. However, as in several other nocturnal arthropods, Megalopta's photoreceptors possess a much greater gain of transduction, indicating that nocturnal photoreceptors trade information capacity for sensitivity. By sacrificing photoreceptor signal-to-noise ratio and information capacity in dim light for an increased gain and, thus, an increased sensitivity, this strategy can benefit nocturnal insects that use neural summation to improve visual reliability at night.

Eye ultrastructure in the pollen-feeding beetle, Xanthochroa luteipennis (Coleoptera: Cucujiformia: Oedemeridae)

The pollen-consuming beetle Xanthochroa luteipennis, which belongs to the family Oedemeridae, possesses a nearly spherical eye of approximately 400 microm in diameter. The eye contains 750-800, mostly hexagonal ommatidia, which are of the acone apposition type and have an open rhabdom. A well-developed pupil mechanism controls the light flux to the rhabdom. The pupil is formed with the help of screening pigment translocations, involving primary and secondary (accessory) pigment cells. Cross-sections of rhabdoms reveal that they are developed as ring-like structures, made up of the rhabdomeres of six retinula cells, surrounding a rod-like inner column of two fused rhabdomeres. Rhabdoms of ommatidia in the middle of the eye differ somewhat from those in more peripheral areas. In the former the central rhabdom is circular in cross-section, while in the latter it is spindle-shaped. The rhabdom organization in combination with the distal pupil mechanism is seen as an adaptation to maximize photon capture under a variety of ambient light intensities, for Oedemerid beetles are commonly active during the day as well as the night.

Angular and spectral sensitivity of fly photoreceptors. III. Dependence on the pupil mechanism in the blowfly Calliphora.

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 photoreceptor's 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.

Analyzing the reflections from single ommatidia in the butterfly compound eye with Voronoi diagrams

This paper presents a robust method for the automated segmentation and quantitative measurement of reflections from single ommatidia in the butterfly compound eye. Digital pictures of the butterfly eye shine recorded with a digital camera are processed to yield binary images from which single facet centers can be extracted using a morphological image analysis procedure. The location of the facet centers is corrected by fitting in-line facet centers to a second-order polynomial. Based on the new centers a Voronoi diagram is constructed. In the case of the eye shine images, the Voronoi diagram defines a hexagonal lattice that overlaps with the original facet borders, allowing instantaneous quantification of the reflections from single ommatidia. We provide two typical examples to demonstrate that the developed technique may be a powerful tool to characterize in vivo the heterogeneity of butterfly eyes and to study the dynamic control of the light flux by the pupil mechanism.

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.

Relationships between pupil working range and habitat luminance in flies and butterflies

The luminance range over which the pupil mechanism operates was measured with pupil reflectometry in 11 species of butterflies and 13 species of dipteran flies. The different species were selected to be as different as possible regarding the range of ambient luminances in which they are active. Habitat luminance ranges were also measured and correlated to luminances in the experimental situation. The pupil mechanism in butterflies operates in the centre of the luminance range in which the different species are active. Three distinct groups of butterflies with pupil sensitivities matched to their specific types of activity pattern were identified: species active only in direct sunlight, species active also in shaded places and species extending their activity into dawn and dusk. Quite differently, the pupil mechanisms of dipteran flies operate in the upper end of the ambient luminances, and in some species well above the luminances normally encountered by the animal. All fly pupils start to close roughly at the same luminance, irrespective of the luminances in which the species are active. The results suggest that the most important role for the pupil mechanism in many of the butterfly species is to maximize acuity over a wide range of luminances, whereas in flies it is to avoid saturation of transduction units and thereby maximize the photoreceptor's signal-to-noise ratio at high light intensities.

Differential effects of ninaC proteins (p132 and p174) on light-activated currents and pupil mechanism in Drosophila photoreceptors.

The Drosophila ninaC locus encodes two retinal specific proteins (p132 and p174) both consisting of a protein kinase joined to a myosin head domain and a C terminal with a calmodulin-binding domain. The role of p132 and p174 was studied via whole-cell recording and through measurements of the pupil mechanism, i.e. the pigment migration in the photoreceptor cells, in the ninaC mutants, P[ninaC delta 132] (p132 absent), P[ninaC delta 174] (p174 absent), and ninaCP235 (null mutant). Voltage-clamped flash responses in P[ninaC delta 174] and ninaCP235 showed delayed response termination. In response to steady light, plateau responses in both P[ninaC delta 174] and ninaCP235 were also large. In both cases the defect was significantly more severe in ninaCP235. Responses in P[ninaC delta 132] were apparently normal. P[ninaC delta 174] and ninaCP235 were also characterized by spontaneous quantum bump-like activity in the dark and by larger and longer light-induced quantum bumps. The turn-off of the pupil mechanism in P[ninaC delta 174] and ninaCP235 was also defective, although in this case the rate of return to baseline in both mutants was more or less the same. In all ninaC mutants, the amplitudes of the pupillary pigment migration were distinctly smaller than that in the wild type. The reduction of the amplitude was largest in P[ninaC delta 174]. The light sensitivity of the pupil mechanism of P[ninaC delta 174] compared to that of wild type was reduced by 1.3 log units. Remarkably, the light sensitivity of P[ninaC delta 132] and ninaCP235 was ca. 0.5 log units higher than that of the wild type. The results suggest that the p174 protein is required for normal termination of the transduction cascade. The diverse phenotypes observed may suggest multiple roles calmodulin distribution for controlling response termination and regulating pigment migration in Drosophila photoreceptors.

Calcium homeostasis in photoreceptor cells of Drosophila mutants inaC and trp studied with the pupil mechanism.

The light-driven pupil mechanism, consisting of an assembly of mobile pigment granules inside the photoreceptor cells, has been investigated by in vivo reflection microspectrophotometry in wild type (WT) Drosophila and in the photoreceptor mutants inaC and trp. The pupillary response of a dark-adapted WT eye to a step in light is a monophasic reflectance increase reaching a plateau after ca. 15-s light adaptation. This reflectance change is due to photoreceptor pigment granules that accumulate near the tips of the rhabdomeres under light adaptation and that are withdrawn towards the periphery in the dark (Franceschini & Kirschfeld, 1976). The step response of the pupil mechanism of inaC is triphasic. Strikingly, the reflectance level at light onset is distinctly higher than that in WT, due to a partly aggregated state of the photoreceptor pigment granules near the rhabdomere tips that persists in the dark-adapted state, in line with direct calcium measurements of Peretz et al. (1994b). The step response of the pupil mechanism of inaC is slightly elevated compared to that of WT. The step response in trp is a transient, biphasic reflectance change, approximating a log normal function. This function is also a good approximation of the pulse response in WT and inaC. The intensity range of pupillary sensitivity is about 4 log unit. The range of inaC compared to that of WT is slightly (approximately 0.5 log unit) shifted towards lower intensities, but that in trp is strongly shifted to higher intensities (approximately 2.5 log unit). The results can be interpreted with the present knowledge of the primary steps in fly phototransduction and the hypothesis that the local intracellular calcium concentration determines the position of the pigment granules, and hence are in line with the notion that the pupil can be used as a qualitative Ca2+ probe.

Pupil adjustments in the eye of the common backswimmer

The pupil mechanism in the acone apposition eye of the semi-aquatic common backswimmer Notonecta glauca (Hemiptera) was investigated with infrared reflectometry of the pseudopupil. This method allows non-invasive continuous measurements of pupil responses in the living animal. The dynamic range of the pupil sensitivity is about 7 log units during daytime and 6 log units at night. During the day, the sensitivity range of the pupil covers the normal daylight intensities in the animal's habitat, just under the water surface (I50=10(19.2) photons m-2 sr-1 s-1). At night, the sensitivity is 1 log unit lower (I50=10(20.2) photons m-2 sr-1 s-1), ensuring that the pupil is maximally open when light intensities are low. During daytime, light adaptation is completed in slightly less than 40 min, and dark adaptation takes approximately 50 min. The pupil response is only slightly slower at night. The speed of the response as well as the pupil sensitivity are dependent on the preceding adaptation history. An endogenous circadian rhythm determines the control range of the pupil aperture. However, the rhythm is easily disturbed, especially within a 3 h period before dusk and dawn. The results are compared with corresponding results from other insects with the same type of pupil mechanism.

Circadian and light-dependent control of the pupil mechanism in tipulid flies

Infrared reflectometry of the deep pseudopupil was used for measuring the absolute sensitivity and the dynamics of the pupil mechanism in the acone apposition eye of tipulid flies. The following species were studied: Tipula luteipennis, T. fusca, T. pagana, T. paludosa and T. staegeri. Pupil responses plotted against light intensity produced S-shaped R logI curves that were fitted by Lipetz equations with I 50-values (intensity corresponding to 50% of the maximal response) ranging between 10 17.4 and 10 20.2 photons m −2 str −1 s −1. The sensitivity of the pupil is considerably higher (1–2 log units) during daytime than at night in all examined species. Differences in the sensitivity between different tipulid species are explained by the different light intensities in their natural environment. The dynamic range of the pupil mechanism is exceptionally large. In T. luteipennis, the pupil operates over a total range of about 8.5 log units of light intensity. This implies that the pupil mechanism of T. luteipennis is functional at light intensities ranging from bright sunlight down to moonlight. The speed of the pupillary movements are slower at night than during the day. During daytime, light adaptation is completed in about 30 min and dark adaptation in about 50 min. At night, the corresponding values are 45 and 60 min. The differences in speed and sensitivity of the pupil mechanism between day and night are due to a circadian rhythm, setting the control range of the pupil aperture.

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.

Sensitivity and dynamics of the pupil mechanism in two tenebrionid beetles

Using infrared reflectometry of the deep pseudopupil, we have measured the absolute sensitivity, the dynamic range and the speed of the pupil mechanism in the acone apposition eye of two tenebrionid beetles: Zophobas morio F. and Tenebrio molitor L. The following conclusions are made from the results: 1. There is a substantial difference in sensitivity of the pupil mechanism between the two beetle species. The pupil is about 5.3 log units more sensitive in Zophobas than in Tenebrio. 2. There is also a difference in sensitivity between day and night. Surprisingly, the sensitivity is higher at day-time, and the difference is about 0.5 log units in both Zophobas and Tenebrio. 3. Light adaptation is completed faster during daytime than at night in both Zophobas and Tenebrio, whereas dark adaptation is completed about equally fast both day and night in both species. The speed of the pupil response, however, is dependent on the preceding adaptation history. 4. The pupil mechanism in both species is under the influence of a circadian rhythm, which determines the size of the pupil aperture in such a way that the pupil is maximally open when dark-adapted at night, but only partially open when dark-adapted at daytime.

Waveguide modes and pupil action in the eyes of butterflies

The rhabdoms of the eyes of butterflies can support one, two, or more waveguide modes, depending on their diameter. The pupil mechanism consists of pigment granules that migrate towards the rhabdom in the light, and absorb the modal light that travels outside the rhabdom. Because there is relatively more extra-rhabdomal light in the higher-order modes, these are shed earlier and at lower light intensities, than the first mode. The effect of this is to reduce the acceptance angle of each ommatidium. We have measured the extent and timecourse of this change by both optical and electrophysiological methods. In small butterflies whose rhabdoms support only one mode there is no change. In Nymphalids such as Vanessa itea, where there are two modes, the reduction is by ca. 30% and in the crepuscular Satyrid Melanitis leda where three or more modes are present the reduction is almost 50%, from 2.9 degrees to 1.5 degrees. These pupil-induced acuity changes will occur towards the lower end of the range of environmental luminances seen by the butterfly during natural flight.

Insect pupil mechanisms

The spectral characteristics of the pupil mechanism in blowfly photoreceptors and their dependence on light intensity have been investigated together with the intensity dependence of the receptor potential. The threshold for the pupil response as measured by reflectance is found at an intensity at which the peak of the receptor potential is about half maximal and the plateau potential starts to saturate. The reflectance saturates at about 3 log-units above threshold. The reflectance spectrum peaks near 620 nm, and its shape is independent of adaptation intensity. The absorbance change, measured by transmission, is extreme in the blue, at about 470 nm. The shape of the absorbance spectrum is slightly intensity dependent, presumably due to optical waveguide effects. The dynamic ranges of the light-induced reflectance and absorbance changes do not coincide. The reflectance change shows saturation at least 1 to 1.5 log units before the absorbance change saturates.

On the effective optical density of the pupil mechanism in fly photoreceptors

A simple electrophysiological method is described for determining the effective optical density of the intracellular pupil mechanism of insect photoreceptor cells. The method depends on the fact that the photoreceptors can not only be illuminated in the normal, orthodromic way, but also antidromically, i.e. from the back. Two assumptions are made: (i) the receptor potential generating process is invariant for the direction of the incident light; (ii) the pupil mechanism is locally driven and only active distally in the cell. The method was tested in the blowfly Calliphora erythrocephala. The two assumptions appeared to be valid there. The electrophysiologically estimated effective optical density of the pupil was equal to the optically measured absorbance over the investigated two log-unit range.

The intracellular pupil mechanism and photoreceptor signal: noise ratios in the fly Lucilia cuprina

The function of the intracellular pupil mechanism is examined by comparing the responses of photoreceptors in normal flies with those from white-eyed flies that lack the pupil. In white-eyed flies the response to an intensity increment of fixed contrast decreases at high background intensities. There is a smaller decrease in noise amplitude so that the signal:noise ratio falls. The intensity dependence of the photoreceptor signal:noise ratio fits a simple model in which activated photopigment molecules compete for 3 X 10(4) transduction units. The signal:noise ratio decreases at high intensities because the transduction units are saturated. This model is supported by a noise analysis, which provides three estimates of the number of events generating photoreceptor responses. In white-eyed flies the event number saturates at high background intensities, suggesting that a maximum of 2 X 10(4) events can be simultaneously active. Wild-type flies do not exhibit saturation effects over the range of intensities studied. The signal:noise ratio rises with intensity to reach a stable asymptote, close to the maximum observed for white-eyed flies. Pupil attenuation is calculated from measurements of signal:noise ratio in white-eyed and wild-type flies. The pupil is progressively activated over a two log unit intensity range and when fully closed attenuates the effective intensity by 99%. The threshold of this pupil effect coincides with the threshold of pupil activation measured optically. We conclude that the intracellular pupil attenuates the light flux to prevent receptor saturation and to extend the range of intensities at which fly photoreceptors operate close to their maximum signal:noise ratio. This upper limit is determined by the number of transduction units generating a cell's response.

Fluorescence of blowfly metarhodopsin

The visual pigment of blowfly peripheral photoreceptors displays a marked red fluorescence when the pigment is in the metarhodopsin state as demonstrated in vivo by simultaneous measurements of transmission and fluorescence. The rhodopsin state is non-fluorescent.It is argued that fluorescence offers a unique means to study visual pigment properties in completely intact living animals and, furthermore, provides the opportunity for studying the photoreceptor optics of visual waveguides. As an example the action of the pupil mechanism of blowfly visual sense cells on the fluorescence signal is demonstrated.

Insect pupil mechanisms

The light-dependent pigment migration system of dragonfly ocelli was studied by optical, non-invasive techniques. The median ocellus is comprised of two lateral halves, as can be demonstrated in the intact animal since illumination of the receptors in one half of the median ocellus only induces a movement of pigment located in that half. Measurable pigment migration can occur within a few seconds, but its speed and extent depends on light intensity. Dispersal of pigment, which occurs upon light adaptation, proceeds faster than retraction, which occurs upon dark adaptation. Action spectra for pigment movement have been determined in Sympetrum and Anax. The spectrum for Sympetrum has a prominent UV peak, moderate blue sensitivity, and very low green sensitivity. A similar profile is obtained in Anax, but only after intense orange adaptation which suppresses the green sensitivity. The results conform to the known spectral sensitivities of Libellulid and Aeschnid ocellar receptors. It is concluded that the photoreceptors drive pigment movement through an unknown mechanism. The effect of the migration of pigment is the selective reduction of radiant flux on the retina from luminous sources at high elevations relative to the animal's normal flying posture.